Interleukin-9 mutein peptides

ABSTRACT

A C to T DNA variation at position 3365 in exon 5 of the human Asthma Associated Factor 1 (AAF1) produces the predicted amino acid substitution of a methionine for a threonine at codon 117 of AAF1. When this substitution occurs in both alleles in one individual, it is associated with less evidence of atopic allergy including asthma, fewer abnormal skin test responses, and a lower serum total IgE. Thus, applicant has identified the existence of a non-asthmatic, non-atopic phenotype characterized by methionine at codon 117 when it occurs in both AAF1 gene products in one individual.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 09/848,585 (filed May 4, 2001) now U.S. Pat. No. 6,645,492, which isa continuation application of U.S. application Ser. No. 09/325,571(filed Jun. 4, 1999), now U.S. Pat. No. 6,261,559 (issued Jul. 17,2001), which is a continuation application of U.S. application Ser. No.08/874,503 (filed Jun. 13, 1997), now abandoned, which is a divisionalapplication of U.S. application Ser. No. 08/697,419 (filed Aug. 23,1996) now abandoned, which claims the benefit of U.S. ProvisionalApplication 60/002,765 (filed Aug. 24, 1995) all of which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to regulating IL-9 activity and treating atopicallergies and related disorders like asthma, based upon the relationshipbetween IL-9 and its receptor.

BACKGROUND OF THE INVENTION

Inflammation is a complex process in which the body's defense systemcombats foreign entities. While the battle against foreign entities maybe necessary for the body's survival, some defense systems improperlyrespond to foreign entities, even innocuous ones, as dangerous andthereby damage surrounding tissue in the ensuing battle.

Atopic allergy is an ecogenetic disorder, where genetic backgrounddictates the response to environmental stimuli. The disorder isgenerally characterized by an increased ability of lymphocytes toproduce IgE antibodies in response to ubiquitous antigens. Activation ofthe immune system by these antigens leads to allergic inflammation andmay occur after ingestion, penetration through the skin, or afterinhalation. When this immune activation occurs and pulmonaryinflammation ensues this disorder is broadly characterized as asthma.Certain cells are critical to this inflammatory reaction and theyinclude T cells and antigen presenting cells, B cells that produce IgE,and mast cells/basophils and eosinophils that bind IgE. Theseinflammatory cells accumulate at the site of allergic inflammation andthe toxic products they release contribute to the tissue destructionrelated to the disorder.

While asthma is generally defined as an inflammatory disorder of theairways, clinical symptoms arise from intermittent air flow obstruction.It is a chronic disabling disorder that appears to be increasing inprevalence and severity¹. It is estimated that 30–40% of the populationsuffer with atopic allergy, and 15% of children and 5% of adults in thepopulation suffer from asthma.¹ Thus, an enormous burden is placed onour health care resources.

The mechanism of susceptibility to atopy and asthma remains unknown.Interestingly, while most individuals experience similar environmentalexposures, only certain individuals develop atopic allergy and asthma.This hypersensitivity to environmental allergens known as “atopy” isoften indicated by elevated serum IgE levels or abnormally great skintest response to allergens in atopic individuals as compared tononatopics.¹⁰ Strong evidence for a close relationship between atopicallergy and asthma is derived from the fact that most asthmatics haveclinical and serologic evidence of atopy.⁴⁻⁹ In particular, youngerasthmatics have a high incidence of atopy.¹⁰ In addition, immunologicfactors associated with an increase in serum total IgE levels are veryclosely related to impaired pulmonary function.³

Both the diagnosis and treatment of these disorders are problematic.¹The assessment of inflamed lung tissue is often difficult, andfrequently the source of the inflammation cannot be determined. Withoutknowledge of the source of the airway inflammation and protection fromthe inciting foreign environmental agent or agents, the inflammatoryprocess cannot be interrupted. It is now generally accepted that failureto control the pulmonary inflammation leads to significant loss of lungfunction over time.

Current treatments suffer their own set of disadvantages. The maintherapeutic agents, beta agonists, reduce the symptoms, i.e.,transiently improve pulmonary functions, but do not affect theunderlying inflammation so that lung tissue remains in jeopardy. Inaddition, constant use of beta agonists results in desensitization whichreduces their efficacy and safety.² The agents that can diminish theunderlying inflammation, the anti-inflammatory steroids, have their ownknown list of disadvantages that range from immunosuppression to boneloss.²

Because of the problems associated with conventional therapies,alternative treatment strategies have been evaluated.⁶⁵⁻⁶⁶ GlycophorinA,⁶⁴ cyclosporin,⁶⁵ and a nonapeptide fragment of IL-2,⁶³ all inhibitinterleukin-2 dependent T lymphocyte proliferation and therefore, IL-9production,⁵¹ however, they are known to have many other effects. Forexample, cyclosporin is used as a immunosuppressant after organtransplantation. While these agents may represent alternatives tosteroids in the treatment of asthmatics,⁶³⁻⁶⁶ they inhibit interleukin-2dependent T lymphocyte proliferation and potentially critical immunefunctions associated with homeostasis. What is needed in the art is theidentification of a pathway critical to the development of asthma thatexplains the episodic nature of the disorder and the close associationwith allergy that is downstream of these critical immune functions.Nature demonstrated that this pathway is the appropriate target fortherapy since biologic variability normally exists at this pathway andthese individuals are otherwise generally not immunocompromised or illexcept for their symptoms of atopy.

Because of the difficulties related to the diagnosis and treatment ofasthma, the complex pathophysiology of this disorder is under intensivestudy. Although this disorder is heterogeneous and may be difficult todefine because it can take many forms, certain features are found incommon among asthmatics. Examples of such traits include elevated serumIgE levels, abnormal skin test response to allergen challenge, bronchialhyperresponsiveness (BHR), bronchodilator reversibility, and airflowobstruction.³⁻¹⁰ These expressions of these asthma related phenotypesmay be studied as quantitative or qualitative measures.

Elevated IgE levels are also closely correlated with BHR, a heightenedbronchoconstrictor response to a variety of stimuli.^(4,6,8,9) BHR isbelieved to reflect the presence of airway inflammation,^(6,8) and isconsidered a risk factor for asthma.¹¹⁻¹² BHR is accompanied bybronchial inflammation and an allergic diathesis in asthmaticindividuals.¹³⁻²¹ Even in children with no symptoms of atopy and asthma,BHR is strongly associated with elevated IgE levels.¹⁹

A number of studies document a heritable component to atopy andasthma.^(4,10,21) However, family studies have been difficult tointerpret since these disorders are significantly influenced by age andgender, as well as many environmental factors such as allergens, viralinfections, and pollutants.²²⁻²⁴ Moreover, because there is no knownbiochemical defect associated with susceptibility to these disorders,the mutant genes and their abnormal gene products can only be recognizedby the anomalous phenotypes they produce. Thus, an important first stepin isolating and characterizing a heritable component is identifying thechromosomal locations of the genes.

Cookson et al. provided the first description of a genetic localizationfor inherited atopy.²⁵ These investigators described evidence forgenetic linkage between atopy and a single marker on a specificchromosomal region designated 11q13.1. Later, they suggested evidence ofmaternal inheritance for atopy at this locus.²⁶ Although maternalinheritance (genetic imprinting) had been observed for atopy, it hadnever been explained previously. However, efforts to confirm thislinkage have not been generally successful.²⁷⁻³¹

Recently, the beta subunit of the high-affinity IgE receptor was mappedto chromosome 11q, and a putative mutation associated with atopy hasbeen described in this gene.³²⁻³³ However, because of the difficultiesby others of replicating this linkage, the significance of this gene andpolymorphism remains unclear. While additional studies will be requiredto confirm whether this putative mutation causes atopy in the generalpopulation, data collected so far suggests this polymorphism is unlikelyto represent a frequent cause of atopy.

Because serum IgE levels are so closely associated with the onset andseverity of allergy and asthma as clinical disorders, attention hasfocused on studies of the genetic regulation of serum total IgE levels.While past studies have provided evidence for Mendelian inheritance forserum total IgE levels,³⁴⁻³⁸ an indication of the existence of oneregulatory gene, others have found evidence for polygenic inheritance ofIgE, i.e., existence of several responsible genes.^(⇄)

Artisans have found several genes that may be important in theregulation of IgE and the development or progression of bronchialinflammation associated with asthma on chromosome 5q. They include genesencoding several interleukins, such as IL-3, IL-4, IL-5, IL-9, IL-13,granulocyte macrophage colony stimulating factor (GM CSF), a receptorfor macrophage colony stimulating factor (CSF-1R), fibroblast growthfactor acidic (FGFA), as well as others.⁴⁰ Recent evidence from familystudies suggests genetic linkage between serum IgE levels and DNAmarkers in the region of these candidate genes on chromosome 5q.^(41,42)Together, these investigations suggest that one or more major genes inthe vicinity of the interleukin complex on chromosome 5q regulates asignificant amount of the observed biologic variability in serum IgEthat is likely to be important in the development of atopy and asthma.

Linkage (sib-pair analyses) was also used previously to identify agenetic localization for BHR.⁷⁹ Because BHR was known to be associatedwith a major gene for atopy, chromosomal regions reported to beimportant in the regulation of serum IgE levels were examined.⁴²Candidate regions for atopy have been identified by linkage analyses.These studies identified the existence of a major gene for atopy onhuman chromosome 5q31-q33.⁴²

Therefore, to determine the chromosomal location of a gene(s) providingsusceptibility to BHR, which would be coinherited with a major gene foratopy, experiments were carried out using linkage analyses between BHRand genetic markers on chromosome 5q.^(42,79,82) Individuals with BHRwere identified by responsiveness to histamine. Markers useful formapping asthma-related genes are shown in FIG. 1.

Specifically, gene candidates for asthma, bronchial hyperresponsiveness,and atopy are shown (right) in their approximate location relative tothe markers shown. The map includes the interleukin genes IL-4, IL-13,IL-5, and IL-3; CDC25, cell division cycle-25; CSF2,granulocyte-macrophage colony stimulating factor (GMCSF); EGR1 earlygrowth response gene-1; CD14, cell antigen 14; ADRB2, thebeta-2-adrenergic receptor; GRL1, lymphocyte-specific glucocorticoidreceptor; PDGFR, platelet-derived growth factor receptor. Bands 5q31-q33extend approximately from IL-4 to D5S410. The distances reported aresex-averaged recombination fractions.

Affected sib-pair analyses demonstrated statistically significantevidence for linkage between BHR and D5S436, D5S658, and several othermarkers located nearby on chromosome 5q31-q33.⁷⁹ These data stronglysupported the hypothesis that one or more closely spaced gene(s) onchromosome 5q31-q33 determine susceptibility to BHR, atopy, andasthma.^(79,80,81,82)

Recently linkage has also been demonstrated between the asthma phenotypeand genetic markers on chromosome 5q31-q33.⁸³ This region of the humangenome was evaluated for linkage with asthma because of the large numberof genes representing reasonable positional candidates for providinggenetic susceptibility for atopy and BHR.

Linkage was demonstrated using the methods described above.^(42,83)Specifically, 84 families were analyzed from the Netherlands with bothsib-pair and LODs for markers from this same region of chromosome 5qpreviously shown to be linked to BHR and atopy.^(42,83) An algorithm wasused to categorize obstructive airways disease in the asthmatic probandsand their families. This classification scheme was based, as describedpreviously, on the presence or absence of BHR to histamine, respiratorysymptoms, significant smoking history (>5 pack years), atopy as definedby skin test response, airway obstruction (FEV1% predicted<95% CI) andreversibility to a bronchodilator (>9% predicted).

Evidence was found for linkage between asthma and markers on chromosome5q by affected sib pair analysis (N=10, P<0.05) and by maximumlikelihood analysis with a dominant model for the asthma phenotype.⁸³

Asthma was linked to D5S658 with a maximal LOD of 3.64 at theta=0.03,using a dominant model (class 1 affected, class 2–4 uncertain, class 5unaffected) with a gene frequency of 0.015 (prevalence of 3%). A maximalLOD of 2.71 at theta.=0.0 was observed for D5S470 which is approximately5 cM telomeric, or away from IL-9, relative to D5S436.⁸³

Subsequent to the original filing of this application, IL-9 or a genenearby was suggested as likely to be important use atopy and asthma.⁴³The IL-9 suggestion was based on a strong correlation in a randomlyascertained population between log serum total IgE levels and alleles ofa genetic marker in the IL-9 gene.⁴³ This type of association with oneor more specific alleles of a marker is termed “linkage disequilibrium”,and generally suggests that a nearby gene determines the biologicvariability under study.⁴⁴

The IL-9 gene has been mapped to the q31-q33 region of chromosome5.⁴⁰Only a single copy of the gene is found in the human genome.^(45,46)Structural similarity has been observed for the human and murine IL-9genes.^(45,46) Each gene consists of five exons and four intronsextending across approximately four Kb of DNA. Expression of the geneappears to be restricted to activated T cells.^(45,46)

The functions of IL-9 now extend well beyond those originallyrecognized. While IL-9 serves as a T cell growth factor, this cytokineis also known to mediate the growth of erythroid progenitors, B cells,mast cells, and fetal thymocytes.^(45,46) IL-9 acts synergistically withIL-3 in causing mast cell activation and proliferation. This cytokinealso potentiates the IL-4 induced production of IgE, IgG, and IgM bynormal human B lymphocytes.⁴⁸ IL-9 also potentiates the IL-4 inducedrelease of IgE and IgG1 by murine B lymphocytes.⁴⁹ A critical role forIL-9 in the mucosal inflammatory response to parasitic infection hasalso been demonstrated.^(50,51)

In addition to IL-9, chromosome 5q bears numerous other gene candidatesincluding IL-3, IRF1, EGR1, ITK, GRL1, ADRB2, CSF1R, FGFA, ITGA2, CD14,PDGFR, CDC25, CSF2, IL-4, IL-5, IL-12B, and IL-13. These may all beimportant in atopic allergy and as potential targets for therapeuticdevelopment. Moreover, the art lacks any knowledge regarding how thesequence of IL-9 or the function of IL-9 specifically correlates withatopic allergy, asthma, or bronchial hyperresponsiveness. Without suchknowledge, artisans would not know how or whether to use IL-9 to eitherdiagnose or treat these disorders.

The art does provide that IL-9 is a novel cytokine having an apparentmolecular weight of approximately between 20 to 30 kD as determined bysodium dodecyl sulfate polyacrylamide gel electrophoresis under reducingconditions. It is produced as a 144 amino acid protein, that isprocessed to a 126 amino acid glycoprotein. Yang et al., (1990) 85disclose that the DNA sequence encoding IL-9 comprises approximately 630nucleotides, with approximately 450 nucleotides in the proper readingframe for the protein.

It is also known in the art that multiple protein isoforms may begenerated from a single genetic locus by alternative splicing.Alternative splicing is an efficient mechanism by which multiple proteinisoforms may be generated from a single genetic locus. Alternativesplicing is used in terminally differentiated cells to reversibly modifyprotein expression without changing the genetic content of the cells.These protein isoforms are preferentially express ed in differenttissues or during different states of cell differentiation oractivation. Protein isoforms may have different functions and Alms andWhite have cloned and expressed a naturally occurring splice variant ofIL-4, formed by the omission of exon 2, thus called IL-4-delta-2.⁸⁶ Itwas observed that IL-4-delta-2 inhibits T-cell proliferation induced byIL-4.

However, the art lacks any knowledge about IL-9 protein isoforms whichare formed by deletions of exons 2 and 3 or the regulatory functionsexhibited by these truncated proteins. Specifically, their role inregulating the biological activity, namely, the down-regulation of IL-9expression or activity is unclear. Moreover, the formation of suchisoforms by alternative splicing has not been previously observed orused to provide variants of IL-9 which function as agonists orantagonists of the native cytokine.

The art also lacks any knowledge about the role of the IL-9 receptorwith asthma-related disorders. It is known that IL-9 binds to a specificreceptor expressed on the surface of target cells.^(46,52,53) Thereceptor actually consists of two protein chains: one protein chain,known as the IL-9 receptor, binds specifically with IL-9 and the otherprotein chain is the chain, which is shared in common with the IL-2receptor.⁴⁶ In addition, the human IL-9 receptor cDNA has beencloned.^(46,52,53) This cDNA encodes a 522 amino acid protein whichexhibits significant homology to the murine IL-9 receptor. Theextracellular region of the receptor is highly conserved, with 67%homology existing between the murine and human proteins. The cytoplasmicregion of the receptor is less highly conserved. The human cytoplasmicdomain is much larger than the corresponding region of the murinereceptor.⁴⁶

The IL-9 receptor gene has also been characterized.⁵³ It is thought toexist as a single copy in the mouse genome and is composed of nine exonsand eight introns.⁵³ The human genome contains at least four IL-9receptor pseudogenes. The human IL-9 receptor gene has been mapped tothe 320 kb subtelomeric region of the sex chromosomes X and Y.⁴⁶Nonetheless, despite these studies, the art lacks any knowledge of arelation between the IL-9 receptor and atopic allergy, asthma, orbronchial hyperresponsiveness.

Thus, the art lacks any knowledge of how the IL-9 gene, its receptor,and their functions, are related to atopic allergy, asthma, bronchialhyperresponsiveness, and related disorders. Therefore, there is aspecific need in the art for genetic information on atopic allergy,asthma, bronchial hyperresponsiveness, and for elucidation of the roleof IL-9 in the etiology of these disorders. There is also a need forelucidation of the role of the IL-9 receptor and the IL-9 receptor genein these disorders. Furthermore, most significantly, based on thisknowledge, there is a need for the identification of agents which arecapable of regulating the interaction between IL-9 and its receptor fortreating these disorders.

SUMMARY OF THE INVENTION

Applicant has satisfied the long felt need for a treatment for atopicallergy including asthma and related disorders by providing informationdemonstrating the role of IL-9 (also known as Asthma Associated Factor1, or AAFI) in the pathogenesis of these disorders which information hasled to compounds that are capable of regulating the activity of IL-9.Applicant has also demonstrated conserved linkage and synteny homologiesbetween mice and humans for a gene that determines biologic variabilityin airway hyperresponsiveness. These relationships specifically identifyIL-9 as a gene candidate. In addition, applicant has determined thatIL-9 is critical to a number of antigen-induced responses in miceincluding bronchial hyperresponsiveness, eosinophilia and elevated cellcounts in bronchial lavage, and elevated serum total IgE. These findingstypify the allergic inflammation associated with asthma.

Furthermore, applicant has determined that a C to T nucleic acidvariation at position 3365 in exon 5 of the human IL-9 gene produces thepredicted amino acid substitution of a methionine for a threonine atcodon 117 of IL-9. When this substitution occurs in both alleles in oneindividual, it is associated with less evidence of atopic allergyincluding asthma, fewer abnormal skin test responses, and a lower serumtotal IgE. Thus, applicant has identified the existence of anonasthmatic, nonatopic phenotype characterized by methionine at codon117 when it occurs in both IL-9 gene products in one individual. As anadditional significant corollary, applicant has identified the existenceof susceptibility to an asthmatic, atopic phenotype characterized by athreonine at codon 117. Thus, the invention includes purified andisolated DNA molecules having such a sequence as well as the peptidesencoded by such DNA.

The biological activity of IL-9 results from its binding to the IL-9receptor and the consequent propagation of a regulatory signal inspecific cells. Therefore, IL-9 functions can be interrupted orregulated by the interaction of IL-9 agonists or antagonists with IL-9or its receptor. Down regulation, i.e. reduction of the functionscontrolled by IL-9, is achieved in a number of ways. Administeringagonists or antagonists that can interrupt the binding of IL-9 to itsreceptor is one key mechanism and such agonists and antagonists arewithin the claimed invention. Examples include administration ofpolypeptide products encoded by the DNA sequences of IL-9 or IL-9receptor wherein the DNA sequences contain various mutations. Thesemutations may be point mutations, insertions, deletions, or splicedvariants of IL-9 or its receptor.

A further embodiment of this invention includes the regulation of theactivity of IL-9 by administering “agonists and antagonists.” Theskilled artisan will readily recognize that all molecules containing therequisite 3-dimensional structural conformation and which contain theresidues essential or critical for receptor binding are within the scopeof this invention. Specifically, residues 43–60 and 71–90 of the matureprotein appear to be important for receptor binding. Applicant has shownthat peptides KP-16 (residues 43–60) and KP-20 (residues 71–90) act asreceptor antagonists. In addition, these residues in the native IL-9molecule are predicted to form anti-parallel helical structures. Thethree dimensional structure of the protein suggests that specificallyserine 52 and/or glutamic acid 53 interact with lysine 85, serine 56interacts with lysine 82, and threonine 59 interacts with valine 78. Thethree dimensional coordinates of these anti-parallel helices and therelated functional groups represent the requisite 3-dimensionalconformation critical for receptor binding and compounds which simulatethese relationships are within the scope of this invention.

The biological activity of the IL-9 receptor (also called AsthmaAssociates Factor 2, AAF2) can also be modulated by using soluble IL-9receptor molecules. Such a molecule prevents the binding of IL-9 to thecell-bound receptor and acts as an antagonist for IL-9, and is alsowithin the scope of this invention.

Polyclonal and monoclonal antibodies which block the binding of IL-9 toits receptor are also within the scope of this invention and are usefultherapeutic agents in treating atopic allergy including asthma andrelated disorders.

Another embodiment of this invention relates to the use of isolated DNAsequences containing various mutations such as point mutations,insertions, deletions, or spliced mutations of IL-9 or the IL-9 receptorin gene therapy.

Expression of IL-9 and IL-9 receptor is also down-regulated byadministering an effective amount of synthetic antisense oligonucleotidesequences. The oligonucleotide compounds of the invention bind to themRNA coding for human IL-9 and IL-9 receptor thereby inhibitingexpression of these molecules.

The structure of both IL-9 and the IL-9 receptor have been examined andanalyzed in great detail and amino acid residues of IL-9 critical forreceptor binding have been identified. Based on structural studies andthe binding characteristics of this specific binding pair, thisinvention further includes small molecules tailored such that theirstructural conformation provides the residues essential for blocking theinteraction of IL-9 with the IL-9 receptor. Such blockade results inmodulation of the activity of the receptor and these molecules are,therefore, useful in treating atopic allergies.

Another embodiment of this invention is directed to the regulation ofdownstream signaling pathways necessary for IL-9 function. IL-9 inducestyrosine phosphorylation of Stat3 which appears to be unique to the IL-9signaling pathway⁵⁸ and is useful as a target for inhibitors. Specificand nonspecific inhibitors of tyrosine kinase such as tyrophostins are,therefore, useful in downstream regulation of the physiological activityof IL-9, and are part of the invention.

In a further embodiment aminosterol compounds are also useful intreating atopic allergies and related disorders because they are alsoinvolved in blocking signal transduction of the IL-9 signal transductionpathway.

The products discussed above represent various effective therapeuticagents in treating atopic allergies, asthma and other related disorders.

This invention also includes the truncated polypeptides encoded by theDNA molecules described above. These polypeptides are capable ofregulating the interaction of IL-9 with the IL-9 receptor.

Thus, applicant has identified the critical role of the IL-9 pathway inpathogenesis of atopic allergy, including bronchial hyperresponsiveness,asthma, and related disorders. More specifically, applicant has providedantagonists and methods of identifying antagonists that are capable ofregulating the interaction between IL-9 and its receptor. Applicant alsoprovides methods for regulating the activity of IL-9 by: 1)administering a compound having activity comparable to IL-9 containingmethionine at codon 117 and the ability to bind to a receptor for IL-9in an amount sufficient to down-regulate the activity of IL-9; and 2) byadministering truncated protein products encoded by isolated nucleicacid sequences comprising deletions of any one or more of exons 1, 2, 3,4, or 5.

Having identified the critical role of the IL-9 pathway in atopicallergy, bronchial hyperresponsiveness, and asthma, applicant alsoprovides a method for the diagnosis of susceptibility to atopic allergy,asthma, and related disorders. Lastly, applicant provides a method forassaying the functions of IL-9 and its receptor to identify compounds oragents that may be administered in an amount sufficient to down-regulateeither the expression or functions of IL-9 and the IL-9 receptor.

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and, together with the description, serve to explain theprinciple of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Map showing the relative order and distance in centiMorgans (cM)between the polymorphic genetic markers useful for mappingasthma-related genes.

FIG. 2: Illustration of the genetic map of human chromosome 5q31-q33 andsyntenic regions in the mouse.

FIG. 3: The LOD score curve on mouse chromosome 13 foratracurium-induced airway responsiveness in mice with increasedsusceptibility to bronchoconstrictor stimuli.

FIG. 4: Alignment of amino acid sequences corresponding to exon of thehuman and murine IL-9 genes. The first sequence is translated from theThr allele of the human gene. The middle sequence is translated from theMet allele of the human gene. The final sequence is translated from themurine gene.

FIG. 5: Histogram of the correlation between human IL-9 gene alleles andserum total IgE titers measured in international units. S/S denotesThr/Thr individuals, S/R denotes Thr/Met individuals and R/R denotesMet/Met individuals.

FIG. 6: Illustration the simple sequence repeat polymorphism at the IL-9locus.

FIG. 7: Translated cDNA sequence of Thr117 version of IL-9.

FIG. 8: Translated cDNA sequence of Met117 version of IL-9.

FIG. 9: Map of pFlag expression construct with Thr117.

FIG. 10: Sequence of pFlag expression construct for the Thr117 versionof the cDNA from the region surrounding the site of ligation.

FIG. 11: Map of pFlag expression construct with Met117.

FIG. 12: Sequence of pFlag expression construct for the Met117 versionof the cDNA from the region surrounding the site of ligation.

FIG. 13: Western blot of recombinant IL-9 proteins

FIG. 14: Amino acid sequences for inhibitory peptides.

FIG. 15: Inhibition by KP-16 of IL-9 mediated MO7e proliferation.

FIG. 16: Inhibition by KP-20 of IL-9 mediated MO7e proliferation.

FIG. 17: Inhibition by KP-23 of IL-9 mediated MO7e proliferation.

FIG. 18: Inhibition by various tyrophostins of IL-9 mediated MO7eproliferation.

FIG. 19: Inhibition by various aminosterols of IL-9 mediated MO7eproliferation.

FIGS. 20A and 20B: Characterization of the role of IL-9 in the antigenresponse in vivo.

FIG. 21: Histologic examination of lungs from control, ova challenged,and anti-IL-9 pretreated animals.

FIG. 22: Inhibition of the antigen response in vivo by blockingantibodies to the murine IL-9 receptor.

FIG. 23: Expression of human Met117 IL-9 and Thr117 IL-9.

FIG. 24: Binding of the human recombinant Met117 and Thr117 forms ofIL-9 to a soluble receptor.

FIG. 25: Steady state levels of IL-9 in unstimulated and stimulatedmurine splenocytes.

FIGS. 26A and 26B: An appendix of chemical moieties.

FIG. 27: Aminosterols isolated from the dog fish shark.

DETAILED DESCRIPTION OF THE INVENTION

Applicant has resolved the needs in the art by elucidating an IL-9pathway and compositions that affect that pathway that may be used inthe diagnosis, prevention or treatment of atopic allergy includingasthma and related disorders. Asthma encompasses inflammatory disordersof the airways with reversible airflow obstruction. Atopic allergyrefers to atopy, and related disorders including asthma, bronchialhyperresponsiveness (BHR), rhinitis, urticaria, allergic inflammatorydisorders of the bowel, and various forms of eczema. Atopy is ahypersensitivity to environmental allergens expressed as the elevationof serum total IgE or abnormal skin test responses to allergens ascompared to controls. BHR refers to bronchial hyperresponsiveness, aheightened bronchoconstrictor response to a variety of stimuli.

By analyzing the DNA of families that exhibit asthma-related disorders,applicant has identified a polymorphism in the IL-9 gene that correlateswith the biologic variability of serum total IgE as one measurableexpression of atopy. The IL-9 gene (also known as Asthma AssociatedFactor 1 or AAF1) refers to the genetic locus of interleukin-9, acytokine exhibiting a variety of functions involving the regulation ofhuman myeloid and lymphoid systems. The IL-9 gene of the presentinvention is found in the q31-q33 region of human chromosome 5 andchromosome 13 in the mouse.

By polymorphism, applicant means a change in a specific DNA sequence,termed a “locus”, from the prevailing sequence. In general, a locus isdefined as polymorphic when artisans have identified two or more allelesencompassing that locus and the least common allele exists at afrequency of 1% or more.

The polymorphism of the present invention leads to an amino acidsubstitution at residue 117 of IL-9. Specifically, instead of thehydrophilic amino acid threonine, the IL-9 of the present inventionexhibits the hydrophilic amino acid methionine (Met IL-9). On a geneticlevel, the polymorphism of the present invention is a substitution of athymine residue for a cytosine residue at nucleotide position 3365 inthe human IL-9 gene as it is described by Renauld and colleagues (1990)(GenBank accession numbers M30135 and M30136),⁵⁴ or at the comparablenucleotide position 4244 of the human IL-9 gene sequence reported byKelleher et al., (1991) (GenBank accession number M86593).⁵⁵

Individuals with a threonine (Thr) at amino acid 117 of IL-9 in eitherone or both of their alleles (Thr/Thr or Thr/Met) generally exhibitsusceptibility to an asthmatic or atopic allergic phenotype, and thesegenotypes are characterized by higher mean serum total IgE levels in thepopulations studied. In contrast, those individuals with a methionine(Met) a codon 117 of IL-9 in both alleles (Met/Met) exhibit a lack ofasthma, fewer abnormal skin test responses, and a lower serum total IgE.Thus, the Met/Met genotype of IL-9 appears to protect against asthma oratopic allergy.

Accordingly, the invention provides a purified and isolated DNA moleculecomprising a nucleotide sequence encoding human interleukin 9 containingmethionine at position 117 (Met IL-9), or a fragment thereof. Theinvention also includes degenerate sequence of the DNA as well assequences that are substantially homologous. The source of the IL-9 ofthe invention is human. Alternatively, the DNA or fragment thereof maybe synthesized using methods known in the art. It is also possible toproduce the compound by genetic engineering techniques, by constructingDNA by any accepted technique, cloning the DNA in an expression vehicleand transfecting the vehicle into a cell which will express thecompound. See, for example, the methods set forth in Sambrook et al.,(1985) Molecular Cloning: A Laboratory Manual, 2d ed. Cold Spring HarborLaboratory Press.

Airway hyperresponsiveness is found in virtually all asthmatics and insome strains of inbred mice (DBA/2).⁸⁴ Airway hyperresponsiveness is arisk factor for the development of asthma in humans and is used inanimal models of asthma as a physiologic measure to assess the efficacyof treatment for asthma. These data along with human⁷⁹ and murinegenetic mapping results (see Examples 1 and 2) suggest a critical rolefor the murine IL-9 gene product in the airway response of the mouse. Inparticular, the hyperresponsive DBA/2(D2) mice differ from theC57BL/6(B6) hyporesponsive mice⁸⁴ in their expression of steady statelevels of IL-9 (See Example 14, FIG. 25). Furthermore, pretreatment withblocking antibodies to IL-9/IL-9 receptor can optionally providecomplete protection from antigen induced airway hyperresponsiveness andinflammation in mice demonstrating a critical regulatory role for IL-9in these immune responses. Thus, these data demonstrate that althoughdifferent molecular changes produce biologic variability in airwayresponsiveness in humans and mice, these changes arise in the samegene(s) (IL-9/IL-9R) that regulate this pathway. Taken together, theseobservations confirm the critical role of IL-9 and the IL-9 receptor inairway hyperresponsiveness, asthma, and atopic allergy. Moreover, thesedata demonstrate that agents of the convention, which block theinteraction of IL-9 with its receptor, protect against an antigeninduced response such as those detailed above.

Further evidence defining the critical role of IL-9 in the pathogenesisof atopic allergy, bronchial hyperresponsivenss, asthma, and relateddisorders derives directly from the applicants observation that IL-9 iscritical to a number of antigen induced responses in mice. When thefunctions of IL-9 are down regulated by antibody pretreatment prior toaerosol challenge with antigen, the animals can be completely protectedfrom the antigen induced responses. These responses include: bronchialhyperresponsiveness, eosinophilia and elevated cell counts in bronchiallavage, histologic changes in lung associated with inflammation, andelevated serum total IgE. Thus, the treatment of such responses, whichare critical to the pathogenesis of atopic allergy and whichcharacterize the allergic inflammation associated with asthma, by thedown regulation of the functions of IL-9, are within the scope of thisinvention.

Applicant also teaches the regulation of the activity of IL-9 byadministering “agonists and antagonists” to the IL-9 receptor. Theskilled artisan will readily recognize that all molecules containing therequisite 3-dimensional structural conformation and which contain theresidues essential or critical for receptor binding are within the scopeof this invention. Applicant has shown that peptides KP-16 (IL-9residues 43–60) and KP-20 (IL-9 residues 71–90) (produced using standardpeptide automated synthesis techniques, for example, the AppliedBiosystems Model 431A Peptide Synthesizer) act as IL-9 antagonists.Specifically, applicant demonstrates that residues 43–60 and 71–90 ofthe mature protein appear to be important for receptor binding. Inaddition, these residues include most of exon 4 (amino acids 44–88) andare predicted to form anti-parallel helical structures. The threedimensional structure of the protein suggests that specifically serine52 and/or glutamic acid 53 interact with lysine 85, serine 56 interactswith lysine 82, and threonine 59 interacts with valine 78. The threedimensional coordinates of these parallel helices and the relatedfunctional groups represent the requisite 3-dimensional conformationcritical for receptor binding and compounds that simulate theserelationships are within the scope of this invention.

The demonstration of an IL-9 sequence associated with an asthma-likephenotype, and one associated with the lack of an asthma-like phenotype,indicates that the lungs' inflammatory response to antigen is dependenton IL-9, and therefore, that down regulating the function of IL-9 shouldprotect against the antigen induced response. Furthermore, applicantalso provides methods of diagnosing susceptibility to atopic allergy andrelated disorders and for treating these disorders based on therelationship between IL-9 and its receptor.

A receptor is a soluble or membrane bound component that recognizes andbinds to molecules, and the IL-9 receptor (also known as AsthmaAssociated Factor 2 or AAF2) of the invention is the component thatrecognizes and binds to IL-9. The functions of the IL-9 receptor consistof binding an IL-9-like molecule and propagating its regulatory signalin specific cells.⁵⁷⁻⁶⁰ An interruption of that function will lead to adown regulation, i.e., reduction, of either the expression of IL-9 or ofthe functions controlled by IL-9. Accordingly, by virtue of thisinteraction between IL-9 and the IL-9 receptor, certain functions of theorganism are modulated or controlled. For a general discussion ofreceptors, see Goodman and Gilman's The Pharmacologic Basis ofTherapeutics, 7th Edition, MacMillan Publishing Company).

One diagnostic embodiment involves the recognition of variations in theDNA sequence of IL-9. One method involves the introduction of a nucleicacid molecule (also known as a probe) having a sequence complementary tothe IL-9 of the invention under sufficient hybridizing conditions, aswould be understood by those in the art. In one embodiment, the sequencewill bind specifically to the Met117 IL-9 or to Thr117 IL-9, and inanother embodiment will bind to both Met117 IL-9 and Thr117 IL-9.Another method of recognizing DNA sequence variation associated withthese disorders is direct DNA sequence analysis by multiple methods wellknown in the art.⁷⁷ Another embodiment involves the detection of DNAsequence variation in the IL-9 gene associated with thesedisorders.⁷³⁻⁷⁷ These include the polymerase chain reaction, restrictionfragment length polymorphism (RFLP) analysis and single strandedconformational analysis. In a preferred embodiment, applicant providesspecifically for a method to recognize, on a genetic level, thepolymorphism in IL-9 associated with the Thr and Met alleles using aStyI RFLP as described herein. In other embodiments Nla, Pfim1, PflM1,and Nco1 RFLPs may be used to distinguish these two alleles of IL-9genes.

Another embodiment involves treatment of atopic allergy and relateddisorders. In a preferred embodiment, the applicant provides a method ofadministering a compound having activity comparable to Met IL-9 and theability to bind to an IL-9 receptor in an amount sufficient to downregulate the activity of IL-9. A compound having activity comparable toMet IL-9 is a compound that functions similarly but not necessarilyidentically. Thus, it may bind to the IL-9 receptor but without the samephysiological effects. Examples include amino acid sequences of IL-9containing various point mutations and/or deletions and sequencessubstantially homologous thereto. For example, such a compound mayinterrupt the binding of Thr IL-9 to the IL-9 receptor as measured bytechniques known in the art. The invention also encompasses functionallyeffective fragments of the above amino acid sequences. In one suchtechnique, the Thr IL-9 may be considered a “ligand” for the IL-9receptor, and binding between the two may be assessed by ligand-bindingassays which are well known in the art as set forth in Goodman andGilman's The Pharmacologic Basis of Therapeutics, 7th Edition, MacMillanPublishing Company).

In another embodiment, the compound may resemble the Met allele of IL-9in structure. Thus, such a compound may incorporate a methionine incodon 117 of IL-9 or may incorporate another hydrophobic amino acid.Thus, included within the scope of this invention are IL-9 variantscomprising substitutions of Thr at position 117 by amino acids selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, phenylalanine, tryptophan, and methionine. Alternatively, thecompound of the invention may exist as a fragment of IL-9 with astructural composition similar to Met IL-9. In another embodiment of theinvention, the compound may retain functions comparable to Met IL-9, butmay not resemble Met IL-9 in structure. For example, the composition ofthe compound may include molecules other than amino acids. This exampleis merely illustrative and one of ordinary skill in the art wouldreadily recognize that other substitutions and/or deletion analogs ofIL-9 resulting in effective antagonists are also within the scope ofthis invention. As discussed above all molecules containing therequisite 3-dimensional structural conformation and which contain theresidues essential or critical for receptor binding are within the scopeof this invention.

Specific assays may be based on IL-9's known regulation, in part, of theproliferation of T lymphocytes, IgE synthesis, and release from mastcells.⁵⁴⁻⁶⁰ Another assay involves the ability of human IL-9 tospecifically induce the rapid and transient tyrosine phosphorylation ofmultiple proteins in M07e cells.⁵⁷ Because this response is dependent onthe expression and activation of the IL-9 receptor, it represents asimple method or assay for the characterization of potentially valuablecompounds. The tyrosine phosphorylation of Stat3 transcriptional factorappears to be specifically related to the actions of IL-9,⁵⁸ and thisresponse represents a simple method or assay for the characterization ofcompounds within the invention. Still another method to characterize thefunction of IL-9 and IL-9-like molecules involves the well known murineTS1 clone and the D10 clone available from ATCC used to assess humanIL-9 function with a cellular proliferation assay.⁵⁹

The Met IL-9 that forms a part of the invention may be viewed as a “weakagonist” of the IL-9 receptor. Such weak agonists are another preferredembodiment of the invention. The term agonist, according to thisinvention, includes compounds that mimic at least some of the effects ofendogenous compounds by interacting or binding with a receptor. Agoniststhat interact or bind to the IL-9 receptor on the surface of certaincells initiate a series of biochemical and physiological changes thatare characteristic of this cytokine's actions.^(45-51,54-60) To identifyother weak agonists of the invention, one may test for binding to theIL-9 receptor or for IL-9-like functions as described herein and in thecited literature.^(2,45-51,54-60)

The present invention also includes antagonists of IL-9 and itsreceptor. Antagonists are compounds that are themselves devoid ofpharmacological activity but cause effects by preventing the action ofan agonist. To identify antagonist of the invention, one may test forcompetitive binding with a known agonist or for down-regulation ofIL-9-like functions as described herein and in the citedliterature.^(2,45-51,54-60)

The binding of either the agonist or antagonist may involve all knowntypes of interaction including ionic forces, hydrogen bonding,hydrophobic interactions, van der Waals forces, and covalent bonds. Inmany cases, bonds of multiple types are important in the interaction ofan agonist or antagonist with a receptor.

In a further embodiment, these compounds may be analogs of IL-9. IL-9analogs may be produced by point mutations in the isolated DNA sequencefor the gene, nucleotide substitutions, and/or deletions which can becreated by methods that are all well described in the art.⁶²

This invention also includes spliced variants of IL-9 and disclosesisolated nucleic acid sequences of IL-9, which contain deletions of oneor more of its five exons. The term “spliced variants” as used hereindenotes a purified and isolated DNA molecule encoding human IL-9comprising at least one exon. There is no evidence of naturallyexpressed spliced mutants in the art. Thus, the present inventionprovides an isolated nucleic acid containing exons, 1, 4 and 5 of humanIL-9. Other variants within scope of this invention include sequencescomprising exons 1, 2, 3, 4, and 5; exons 1, 2, 3, and 4; exons 1, 2, 4,and 5 and exons 1, 3, 4, and 5. It must be understood that these exonsmay contain various point mutations.

Specific examples of antagonistic peptides derived from IL-9 includeKP-16 (SEQ. ID NO: 13) and KP-20 (SEQ. ID NO: 14) which are derived fromexon 4. Exon 4 encodes 44 amino acids while the peptides mentioned abovecontain 16 and 20 amino acids respectively and they do not overlap.These peptides exhibit considerable inhibitory activity bothindividually and when assayed in combination. Additionally, KP-23 (SEQID NO: 15) and KP-24 (SEQ ID NO: 16) are derived from exon 5 and alsoexhibit similar activity. Splice variants of IL-9 can be formed bydeletion of any one or more of the IL-9 exons 1 through 5. As shownabove, peptides derived from these exons show regulatory capability and,accordingly, are useful in treating atopic allergies, including asthma.

It is known in the art that, in multienzyme systems, the first orregulatory enzyme can be activated or inhibited by the end product ofthe multi-enzyme system. When the concentration of the end productincreases over the steady state concentration, the end product will actas a specific activator or inhibitor of the regulatory enzyme in thesequence. Such feedback mechanism is also relevant to the IL-9 systemand it is observed that the various polypeptides of this invention arecapable of exerting such activation or inhibitory control on theactivity of the IL-9 receptor and possibly the expression or function ofother cytokines and their receptors that play a role in the pathogenesisof asthma.

The invention also includes modifications of agonists or antagoniststhat can be made using knowledge that is routine to those in this art.For example, the affinity of a compound for a receptor is generallyclosely related to the chemical structure of the compound. Thus,structure-activity relationships may be used to modify the agonists andantagonists of the invention. For example, the techniques ofcrystallography/X-ray diffraction and NMR may be used to makemodifications of the invention.

For example, one can create a three dimensional structure of human IL-9that can be used as a template for building structural models ofdeletion mutants using molecular graphics. These models can then be usedto identify and construct a mutant IL-9 molecule with affinity for theIL-9 receptor comparable to IL-9, but with a lower biologic activity.What is meant by lower biologic activity is 2 to 100,000 fold less thanIL-9, preferably 100 to 1,000 fold less than IL-9.

In still another embodiment, these compounds also may be used as dynamicprobes for receptor structure and to develop receptor antagonists usingIL-9 dependent cell lines.

In addition, this invention also provides compounds that prevent thesynthesis or reduce the biologic stability of IL-9 or the IL-9 receptor.Biologic stability is a measure of the time between the synthesis of themolecule and its degradation. For example, the stability of a protein,peptide or peptide mimetic⁸⁹ therapeutic may be prolonged by usingD-amino acids, or shortened by altering its sequence to make it moresusceptible to enzymatic degradation.

In another embodiment, the agonists and antagonists of the invention areantibodies to IL-9 and the IL-9 receptor. The antibodies to IL-9 and itsreceptor may be either monoclonal or polyclonal made using standardtechniques well known in the art (See Harlow & Lane (1988) Antibodies—ALaboratory Manual, Cold Spring Harbor Laboratory Press). They can beused to block IL-9 from binding to the receptor. In one embodiment theantibodies interact with IL-9. In another embodiment the antibodiesinteract with the IL-9 receptor. The IL-9 used to elicit theseantibodies can be any of the IL-9 variants discussed above.

Antibodies are also produced from peptide sequences of IL-9 or the IL-9receptor using standard techniques in the art (see Protocols inImmunology, Chapt. 9, Wiley). The peptide sequences from the murine IL-9receptor that can be used to produce blocking antisera have beenidentified as: GGQKAGAFTC (residues 1–10)(SEQ ID NO: 19);LSNSIYRIDCHWSAPELGQESR (residues 11–32)(SEQ ID NO: 20); andCESYEDKTEGEYYKSHWSEWS (residues 184–203 with a Cys residue added to theN-terminus for coupling the peptide to the carrier protein)(SEQ ID NO:21). In addition, an epitope that binds to a blocking antibody directedto the human IL-9 receptor has been identified as residues 8–14 of themature human IL-9 receptor. (TCLTNNI)(SEQ ID NO: 22) and two epitopesthat bind to blocking antibodies directed to human IL-9 have also beenidentified as residues 50–67 (CFSERLSQMTNTTMQTRY) (SEQ ID NO: 17) andresidues 99–116 (TAGNALTFLKSLLEIFQK) (SEQ ID NO: 16) The human epitopesas well as the human peptides that correspond to the peptides thatproduce blocking antibodies in the murine sequences are most likely tobe useful for the production of therapeutic antibodies.

In still another embodiment, the compounds of the invention may becoupled to chemical moieties, including proteins that alter thefunctions or regulation of the IL-9 pathway for therapeutic benefit inatopic allergy and asthma.⁶¹ These proteins may include in combinationother cytokines and growth factors including⁶⁷ IL-4, IL-5, IL-3, IL-2,IL-13, and IL-10 that may offer additional therapeutic benefit in atopicallergy and asthma. In addition, the IL-9 of the invention may also beconjugated through phosphorylation and conjugated to biotinylate,thioate, acetylate, iodinate, and any of the crosslinking reagents shownin FIGS. 26A and B (Pierce).

In a further embodiment, the invention includes the down regulation ofIL-9 expression or function by administering soluble IL-9 receptormolecules that bind IL-9. Renauld et al., (1992)₅₉ have shown theexistence of a soluble form of the IL-9 receptor. This molecule can beused to prevent the binding of IL-9 to cell bound receptor and act as anantagonist of IL-9. Soluble receptors have been used to bind cytokinesor other ligands to regulate their function.⁸⁷ A soluble receptor is aform of a membrane bound receptor that occurs in solution, or outside ofthe membrane. Soluble receptors may occur because the segment of themolecule which commonly associates with the membrane is absent. Thissegment is commonly referred to in the art as the transmembrane domainof the gene, or membrane binding segment of the protein. Thus, in oneembodiment of the invention, a soluble receptor may represent a fragmentor an analog of a membrane bound receptor. In another embodiment of theinvention, the structure of the segment that associates with themembrane may be modified (e.g. DNA sequence polymorphism or mutation inthe gene) so the receptor is not inserted into the membrane, or thereceptor is inserted, but is not retained within the membrane. Thus, asoluble receptor, in contrast to the corresponding membrane bound form,differs in one or more segments of the gene or receptor protein that areimportant to its association with the membrane.^(52,53)

These compounds may be known forms of a soluble IL-9 receptor that actto bind IL-9. Alternatively, these compounds may resemble known forms ofthe IL-9 receptor, but may exist as fragments. In another embodiment ofthe invention, the compound may retain functions comparable to solubleIL-9 receptor, but may not resemble soluble IL-9 receptor incomposition. For example, the composition of the compound may includemolecules other than amino acids. Thus, these compounds will bind IL-9and prevent IL-9 from acting at its cell surface receptor.

A further embodiment of the invention relates to antisense or genetherapy. It is now known in the art that altered DNA molecules can betailored to provide a specific selected effect, when provided asantisense or gene therapy. The native DNA segment coding for IL-9receptor, has, as do all other mammalian DNA strands, two strands; asense strand and an antisense strand held together by hydrogen bonding.The mRNA coding for the receptor has a nucleotide sequence identical tothe sense strand, with the expected substitution of thymidine byuridine. Thus, based upon the knowledge of the receptor sequence,synthetic oligonucleotides can be synthesized. These oligonucleotidescan bind to the DNA and RNA coding for the receptor. The activefragments of the invention, which are complementary to mRNA and thecoding strand of DNA, are usually at least about 15 nucleotides, moreusually at least 20 nucleotides, preferably 30 nucleotides and morepreferably may be 50 nucleotides or more. The binding strength betweenthe sense and antisense strands is dependent upon the total hydrogenbonds. Therefore, based upon the total number of bases in the mRNA, theoptimal length of the oligonucleotide sequence may be easily calculatedby the skilled artisan.

The sequence may be complementary to any portion of the sequence of themRNA, i.e., it may be proximal to the 5′-terminus or capping site, ordownstream from the capping site, between the capping site and theinitiation codon and may cover all or only a portion of the non-codingregion or the coding region. The particular site(s) to which theantisense sequence binds will vary depending upon the degree ofinhibition desired, the uniqueness of the sequence, the stability of theantisense sequence, etc.

In the practice of the invention, expression of the IL-9 receptor isdown-regulated by administering an effective amount of syntheticantisense oligonucleotide sequences described above. The oligonucleotidecompounds of the invention bind to the mRNA coding for human IL-9 orIL-9 receptors thereby inhibiting expression (translation) of theseproteins (see Gruss et al., (1992) Cancer Res. 52, 1026–1031).

The isolated DNA sequences containing various mutations such as pointmutations, insertions, deletions, or spliced mutations of IL-9 areuseful in gene therapy as well.

In addition to the direct regulation of the IL-9 receptor, thisinvention also encompasses methods of downstream regulation whichinvolve inhibition of signal transduction. In particular, a furtherembodiment of this invention is drawn to inhibition of tyrosinephosphorylation. It is known in the art that highly exergonicphosphoryl-transfer reactions are catalyzed by various enzymes known askinases. In other words, a kinase transfers phosphoryl groups betweenATP and a metabolite. IL-9 induces tyrosine phosphorylation of multipleproteins; it is known in the art that in addition to the activation ofJAK1 and JAK3 tyrosine kinases, IL-9 also induces tyrosinephosphorylation of Stat3.⁵⁸ Phosphorylation of Stat3 is unique to theIL-9 signal transduction pathway and hence is a perfect target forinhibitors.⁵⁸ This invention includes within its scope tyrphostins whichare specific inhibitors of protein tyrosine kinases. Thus, tyrphostins(obtained for example from Calbiochem) and other similar inhibitors ofthese kinases are useful in the modulation of signal transduction andare useful in the treatment of atopic allergies and asthma.

In still another aspect of the invention, it was surprisingly found thataminosterol compounds are also useful in the inhibition of signaltransduction due to IL-9 stimulation. Aminosterol compounds which areuseful in this invention are described in U.S. Pat. No. 5,637,691 andrelated U.S. Pat. No. 5,733,899 and 5,721,226, as well as in U.S. Pat.No. 5,840,740 and related U.S. Pat. Nos. 5,795,885; 5,994,336;5,763,430; 5,840,936; 5,874,597; 5,792,635 and 5,847,172 which arespecifically incorporated herein by reference.

In addition, the invention includes pharmaceutical compositionscomprising the compounds of the invention together with apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectionable solutions. Suitable pharmaceutical carriers are describedin Remington's Pharmaceutical Sciences, specifically incorporated hereinby reference.

The compounds used in the method of treatment of this invention may beadministered systemically or topically, depending on such considerationsas the condition to be treated, need for site-specific treatment,quantity of drug to be administered, and similar considerations.

Topical administration may be used. Any common topical formation such asa solution, suspension, gel, ointment, or salve and the like may beemployed. Preparation of such topical formulations as are well describedin the art of pharmaceutical formulations as exemplified, for example,by Remington's Pharmaceutical Science, Edition 17, Mack PublishingCompany, Easton, Pa. For topical application, these compounds could alsobe administered as a powder or spray, particularly in aerosol form. Theactive ingredient may be administered in pharmaceutical compositionsadapted for systemic administration. As is known, if a drug is to be aadministered systemically, it may be confected as a powder, pill,tablets or the like, or as a syrup or elixir for oral administration.For intravenous, intraperitoneal or intra-lesional administration, thecompound will be prepared as a solution or suspension capable of beingadministered by injection. In certain cases, it may be useful toformulate these compounds in suppository form or as an extended releaseformulation for deposit under the skin or intermuscular injection. In apreferred embodiment, the compounds of this invention be administered byinhalation. For inhalation therapy the compound may be in a solutionuseful for administration by metered dose inhalers, or in a formsuitable for a dry powder inhaler.

An effective amount is that amount which will down regulate either theexpression of IL-9 or the functions controlled by IL-9. A giveneffective amount will vary from condition to condition and in certaininstances may vary with the severity of the condition being treated andthe patient's susceptibility to treatment. Accordingly, a giveneffective amount will be best determined at the time and place throughroutine experimentation. However, it is anticipated that in thetreatment of asthma-related disorders in accordance with the presentinvention, a formulation containing between 0.001 and 5 percent byweight, preferably about 0.01 to 1%, will usually constitute atherapeutically effective amount. When administered systemically, anamount between 0.01 and 100 mg per kg body weight per day, butpreferably about 0.1 to 10 mg/kg, will effect a therapeutic result inmost instances.

Applicant also provides for a method to screen for the compounds thatdown regulate the expression of IL-9 or the functions controlled byIL-9. One may determine whether the functions expressed by IL-9 aredown-regulated using techniques standard in the art.⁵⁷⁻⁶⁰ In a specificembodiment, applicant provides for a method of identifying compoundswith functions comparable to Met IL-9. Thus, in one embodiment, serumtotal IgE may be measured using techniques well known in the art⁴² toassess the efficacy of a compound in down regulating the functions ofIL-9 in vivo. In another embodiment, bronchial hyperresponsiveness,bronchoalveolar lavage, and eosinophilia may be measured usingtechniques well known in the art⁴² to assess the efficacy of a compoundin down regulating the functions of IL-9 in vivo. In yet anotherembodiment, the functions of IL-9 may be assessed in vitro. As is knownto those in the art, human IL-9 specifically induces the rapid andtransient tyrosine phosphorylation of multiple proteins in MO7e cells.The tyrosine phosphorylation of Stat3 transcriptional factor appears tobe specifically related to the actions of IL-9. Another method tocharacterize the function of IL-9 and IL-9-like molecules that dependson the “stable expression” of the IL-9 receptor uses the well knownmurine TS1 clones to assess human IL-9 function with a cellularproliferation assay.⁵¹

The invention also includes a simple screening assay for saturable andspecific ligand binding based on cell lines that express the IL-9receptor.^(46,59) The IL-9 receptor is expressed on a wide variety ofcell types, including K562, C8166-45, B cells, T cells, mast cells,neutrophils, megakaryocytes (UT-7 cells),53 the human megakaryoblasticleukemia cell lines MO7e⁵⁷, TF1,⁵⁹ macrophages, fetal thymocytes, thehuman kidney cell line 293,⁵³ and murine embryonic hippocampalprogenitor cell lines.^(46,52,53) In another embodiment, soluble IL-9receptor may be used to evaluate ligand binding and potential receptorantagonists.

The practice of the present invention will employ the conventional termsand techniques of molecular biology, pharmacology, immunology, andbiochemistry that are within the ordinary skill of those in the art(see, for example, Sambrook et al., (1985) Molecular Cloning: ALaboratory Manual, 2d ed. Cold Spring Harbor Laboratory Press, orAusubel et al., (1994) Current Protocols In Molecular Biology, JohnWiley & Sons).

Nonetheless, we offer the following basic background information. Thebody's genetic material, or DNA, is arranged on 46 chromosomes, whicheach comprises two arms joined by a centromere. Each chromosome isdivided into segments designated p or q. The symbol p is used toidentify the short arm of a chromosome, as measured from the centromereto the nearest telomere. The long arm of a chromosome is designated bythe symbol q. Location on a chromosome is provided by the chromosome'snumber (i.e., chromosome 5) as well as the coordinates of the p or qregion (i.e., q31-q33). In addition, the body bears the sex chromosomes,X and Y. During meiosis, the X and Y chromosomes exchange DNA sequenceinformation in areas known as the pseudoautosomal regions.

DNA, deoxyribonucleic acid, consists of two complementary strands ofnucleotides, which include the four different base compounds, adenine(A), thymine (T), cytosine (C), and guanine (G). A of one strand bondswith T of the other strand while C of one strand bonds to G of the otherto form complementary “base pairs,” each pair having one base in eachstrand.

A sequential grouping of three nucleotides (a “codon”) codes for oneamino acid. Thus, for example, the three nucleotides CAG codes for theamino acid Glutamine. The 20 naturally occurring amino acids, and theirone letter codes, are as follows:

Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp DAsparagine or Asx B Aspartic acid Cysteine Cys C Glutamine Gln QGlutamine Acid Glu E Glutamine or Glx Z Glutamic acid Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

Amino acids comprise proteins. Amino acids may be hydrophilic, i.e.,displaying an affinity for water, or hydrophobic, i.e., having anaversion to water. Thus, the amino acids designated as G, A, V, L, I, P,F, Y, W, C and M are hydrophobic and the amino acids designated as S, Q,K, R, H, D, E, N and T are hydrophilic. In general, the hydrophilic orhydrophobic nature of amino acids affects the folding of a peptidechain, and consequently the three dimensional structure of a protein.

DNA is related to protein as follows:

Genomic DNA comprises all the DNA sequences found in an organism's cell.It is “transcribed” into messenger RNA (“mRNA”). Complementary DNA(“cDNA”) is a complementary copy of mRNA made by reverse transcriptionof mRNA. Unlike genomic DNA, both mRNA and cDNA contain only theprotein-encoding or polypeptide-encoding regions of the DNA, theso-called “exons.” Genomic DNA may also include “introns,” which do notencode proteins.

In fact, eukaryotic genes are discontinuous with proteins encoded bythem, consisting of exons interrupted by introns. After transcriptioninto RNA, the introns are removed by splicing to generate the maturemessenger RNA (mRNA). The splice points between exons are typicallydetermined by consensus sequences that act as signals for the splicingprocess. Splicing consists of a deletion of the intron from the primaryRNA transcript and a joining or fusion of the ends of the remaining RNAon either side of the excised intron. Presence or absence of introns,the composition of introns, and number of introns per gene, may varyamong strains of the same species, and among species having the samebasic functional gene. Although in most cases, introns are assumed to benonessential and benign, their categorization is not absolute. Forexample, an intron of one gene can represent an exon of another. In somecases, alternate or different patterns of splicing can generatedifferent proteins from the same single stretch of DNA. In fact,structural features of introns and the underlying splicing mechanismsform the basis for classification of different kinds of introns.

As to the exons, these can correspond to discrete domains or motifs, asfor example, functional domains, folding regions, or structural elementsof a protein; or to short polypeptide sequences, such as reverse turns,loops, glycosylation signals and other signal sequences, or unstructuredpolypeptide linker regions. The exon modules of the presentcombinatorial method can comprise nucleic acid sequences correspondingto naturally occurring exon sequences or naturally occurring exonsequences which have been mutated (e.g. point mutations, truncations,fusions).

Returning now to the manipulation of DNA, DNA can be cut, spliced, andotherwise manipulated using “restriction enzymes” that cut DNA atcertain known sites and DNA ligases that join DNA. Such techniques arewell known to those of ordinary skill in the art, as set forth in textssuch as Sambrook et al., (1985) Molecular Cloning: A Laboratory Manual,2d ed. Cold Spring Harbor Laboratory Press, or Ausubel et al., (1994)Current Protocols In Molecular Biology, John Wiley & Sons.

DNA of a specific size and sequence can then be inserted into a“replicon,” which is any genetic element, such as a plasmid, cosmid, orvirus, that is capable of replication under its own control. A“recombinant vector” or “expression vector” is a replicon into which aDNA segment is inserted so as to allow for expression of the DNA, i.e.,production of the protein encoded by the DNA. Expression vectors may beconstructed in the laboratory, obtained from other laboratories, orpurchased from commercial sources.

The recombinant vector (known by various terms in the art) may beintroduced into a host by a process generically known as“transformation.” Transformation means the transfer of an exogenous DNAsegment by any of a number of methods, including infection, directuptake, transduction, F-mating, microinjection, or electroporation intoa host cell.

Unicellular host cells, known variously as recombinant host cells,cells, and cell culture, include bacteria, yeast, insect cells, plantcells, mammalian cells and human cells. In particularly preferredembodiments, the host cells include E. coli, Pseudonas, Bacillis,Streptomyces, Yeast, CHO, R1-1, B-W, LH, COS-J, COS-7, BSC1, BSC40,BMT10, and S69 cells. Yeast cells especially include Saccharomtces,Pichia, Candida, Hansenula, and Torulopis.

As those skilled in the art recognize, the expression of the DNA segmentby the host cell requires the appropriate regulatory sequences orelements. The regulatory sequences vary according to the host cellemployed, but include, for example, in prokaryotes, a promoter,ribosomal binding site, and/or a transcription termination site. Ineukaryotes, such regulatory sequences include a promoter and/or atranscription termination site. As those in the art well recognized,expression of the polypeptide may be enhanced, i.e., increased over thestandard levels, by careful selection and placement of these regulatorysequences.

In other embodiments, promoters that may be used include the humancytomegalovierus (CMV) promoter, tetracycline inducible promoter, simianvirus (SV40) promoter, moloney murine leukemia long terminal repeat(LTR) promoter, glucocorticoid inducible murine mammary tumor virus(MMTV) promoter, Herpes thymidine kinase promoter, murine and humanbeta-actin promoters, HTLVl and HIV IL-9 5′ flanking region, human andmouse IL-9 receptor 5′ flanking region, bacterial tac promoter anddrosophila heat shock scaffold attachment region (SAR) enhancerelements.

The DNA may be expressed as a polypeptide of any length such aspeptides, oligopeptides, and proteins. Polypeptides also includetranslational modifications such as glycosylations, acetylations,phosphorylations, and the like.

Another molecular biologic technique of interest to the presentinvention is “linkage analysis.” Linkage analysis is an analytic methodused to identify the chromosome or chromosomal region that correlateswith a trait or disorder.⁴⁴ Chromosomes are the basic units ofinheritance on which genes are organized. In addition to genes, artisanshave identified “DNA markers” on chromosomes. DNA markers are knownsequences of DNA whose identity and sequence can be readily determined.Linkage analysis methodology has been applied to the mapping of diseasegenes, for example, genes relating to susceptibility to asthma, tospecific chromosomes.^(42,44)

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed. It is intended that the specifications and examplesbe considered exemplary only with a true scope of the invention beingindicated by the claims.

EXAMPLES

In conducting the experiments described in the Examples below, applicantused the following methods:

Patient Populations

Asthma families were recruited from two sources.^(27,42,79-63,68) Ineach case patients were genotyped with respect to the polymorphism atposition 3365 of the human IL-9 gene (GenBank accession number M30136).

A third population of 74 individuals was ascertained randomly withrespect to asthma and atopy from the East Coast of the United States.The frequency of the Met substitution at codon 117 was used as anunbiased estimate of the prevalence of this variant in the generalpopulation.

A fourth population of 49 individuals was ascertained randomly withrespect to asthma and atopy from the Philadelphia, Pa. area. Total serumIgE were assayed by enzyme-linked immunosorbent test (ELISA, Genzyme,Cambridge, Mass.). DNA was extracted from the WBC in peripheral bloodfrom each individual. Analyses of genetic markers (genotyping) andcandidate genes were performed on the genomic DNA extracted. Once again,the frequency of the Met substitution at codon 117 was used as anunbiased estimate of the prevalence of this variant in the generalpopulation.

Oligonucleotide Primers.

All primers were designed using OLIGO 4.0. Characterization of the IL-9gene was carried out using primers surrounding each of the 5 exons ofthe reported sequence. The primer sequences surrounding each exon were:exon 1 (upper) (5′ GCT CCA GTC CGC TGT CAA 3′) (SEQ ID NO: 32) and(lower) (5′CTC CCC CTG CAG CCT ACC 3′) (SEQ ID NO: 33) (product size 150bp); exon 2 (upper) (5′ CGG GGC TGA CTA AAG GTT CT 3′) (SEQ ID NO: 34)and (lower) (5′ GTT CTT AAA GAG CAT TCA CT 3′) (SEQ ID NO: 35) (productsize 99 bp); exon 3 (upper) (5′ ATT TTC ACA TCT GGA ATC TTC ACT 3′) (SEQID NO: 36) and (lower) (5′ AAT CCA AGG TCA ACA TTA TG 3′) (SEQ ID NO:37) (product size 113 bp); exon 4 (upper) (5′ TTT CTT TGA ATA AAT CCTTAC 3′) (SEQ ID NO: 38) and (lower) (5′ GAA ATC ACC AAC AGG AAC ATA 3′)(SEQ ID NO: 39) (product size 206 bp); and exon 5 (upper) (5′ ATC AACTTT CAT CCC CAC AGT 3′) (SEQ ID NO: 40) and (lower) (5′ GGA TAA ATA ATATTT CAT CTT CAT 3′) (SEQ ID NO: 41). Each exon was examined first by asingle strand conformational polymorphism assay (SSCP).^(72,77) Theprimers for exon 5 produced a 160 bp product after polymerase chainreaction (PCR) amplification which was also examined by direct solidphase sequence analysis.^(72,77) The upper primer was synthesized with a5′ biotin label and, following amplification, the PCR product wascaptured by a streptavidin-linked paramagnetic bead (Dynal) andcharacterized by Sanger sequencing as described elsewhere.⁷⁷ Sequencepolymorphisms were distinguished from artifact by repeated analyses.

SSCP Analysis.

SSCP, a method for detection of polymorphisms on the basis of changes inmigration of single-stranded DNA exposed to an electric field,⁷² wascarried out as set forth in Schwengel et al., (1994) at room temperaturewith and without 10% glycerol using 6% polyacrylamide gelelectrophoresis at a cross-linking monomer concentration of 2.67%.⁷⁷Four microliters of PCR product was mixed with 5 microliters 2× stopbuffer (95% formamide, 20 mM EDTA, 0.05% BPB, 0.05% xylene cyanol), and1 microliter 0.5% SDS and 50 micromolar EDTA, denatured at 85–90° C. for8 minutes, and then immediately placed on ice. Electrophoresis wascarried out at 12 watts for approximately 24 hours for glycerolcontaining gels and 12 hours for non-glycerol gels. The gels were thendried and exposed to Kodak XAR® film.

DNA Sequencing.

Direct DNA sequencing of the PCR products was accomplished using solidphase techniques after verifying the presence of the correct size PCRproduct on a 1% agarose gel stained with ethidium bromide as set forthin Schwengel et al., (1994).⁷⁷ Twenty microliters of PCR product wasincubated with 40 microliters of Dynabeads® m-280 (Dynal) for 15minutes. The beads were washed and diluted as suggested by themanufacturer. Each sample was subsequently washed with B&W buffercontaining 10 mM tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl, denatured with0.1 N NaOH, and then washed with 0.1 N NaOH, B&W buffer, and 10 mMTris-HCl pH 8 and 1 mM EDTA (TE). The pellet of beads was resuspendedwith 10 microliters of H₂O.

Sanger sequencing reactions were carried out using Sequenase (UnitedStates Biochemical Co.). ³⁵S-DATP or ³³P-DATP was incorporated into thesequencing reactions, and the products were electrophoresed througheither 5% or 6% polyacrylamide gels containing 7 M urea. Gels were driedwithout fixing and exposed to X-ray film. Alleles were determined bycomparing the genotypes of parents and offspring. Infrequent artifactswere easily distinguished from true sequence polymorphisms byrepetition.

DNA was available and extracted from peripheral leukocytes. Genomic DNAwas diluted to a concentration of 200 micrograms/ml foramplification.^(27,42) Simple sequence repeats (SSR) including DXYS154were selected from the Genome Data Base (GDB; Welch library, JohnsHopkins University, Baltimore, Md.). Genotyping of the sKK-1 marker wascarried out using the following primers sKK-1U (5′ CAA ATC TGA AGA GCAAAC TAT 3′) (SEQ ID NO: 42) and sKK-1L (5′ TTA AAA AAT TCA TTT CAG TATTCT 3′) (SEQ ID NO: 43) which produce a 90 bp product. Each SSR productwas amplified by PCR⁷² and sized according to methods previouslydescribed.^(27,42) Sample handling was carried out as described by Weberet al. with minor modifications.^(71,27,42) Genotypes were determinedfrom two independent readings of each autoradiograph. Individualsgenotyping the families were blinded to the clinical data.

RFLP Analysis.

As a result of the C to T polymorphism at position 3365, a StyIrestriction fragment length polymorphism (RFLP) was produced at position52 of the IL-9 exon 5 PCR product. To test for the presence of this DNAsequence variant the lower primer from exon 5 was end-labeled prior toPCR amplification. The PCR product was then digested with StyI producingtwo fragments 108 bp (labeled) and 52 bp (unlabeled) in length. ThisRFLP was used along with SSCP to confirm the presence of thispolymorphism in families and individuals.

Linkage Analyses and Data Management.

Linkage analyses were performed using affected sib-pair methods (SIBPAL,S.A.G.E.),⁷⁸ an established approach for the investigation of thegenetic basis of complex traits, such as BHR, atopy, and asthma.Affected sib-pairs are usually tested first, since a proportion ofunaffected sib-pairs may still be gene carriers but do not express thetrait. In contrast to LOD score methods where the model of inheritance(dominant, recessive, etc.) must be specified exactly, analysis bysib-pair methods makes no explicit assumptions in this regard. Thus, insib-pair analyses the parents' clinical information is not used intesting for linkage. The pertinent observation in these methods is howoften two affected offspring share copies of the same parental markerallele.⁴⁴ If the same copy of a parental marker allele is observed indifferent offspring, they are said to be inherited “identical bydescent.” Linkage is suggested when affected sib-pairs are identical bydescent for a marker allele significantly more often than expected bychance (50%). When the same marker allele is transmitted with thedisease gene in different offspring, this implies that the marker locusis linked, or must be located close enough on the same chromosome, tothe disease gene so they cosegregate during meiosis. The trait is thenmapped by knowing the chromosomal localization of the marker.

Linkage in humans may also be established by the method of likelihoodratios. This method involves comparison of the probability that observedfamily data would arise under one hypothesis, for instance, linkagebetween two DNA markers, to the probability that it would arise under analternative hypothesis, typically, nonlinkage. The ratio of theseprobabilities is called the odds ratio for one hypothesis relative tothe other. By convention, mammalian geneticists prefer the log of theodds ratio, or the LOD score. Generally, linkage is considered provedwhen the odds in favor of linkage versus nonlinkage become overwhelming,or reach 1,000:1 (LOD=3). Linkage is rejected when the odds drop to100:1 against this hypothesis (LOD=−2). The maximum likelihood estimateis the recombination fraction where the likelihood ratio is largest.LODs from multiple pedigrees are thus added until the score grows to 3(signifying 1,000:1 odds) or falls to −2 (indicating 100:1 odds).

All clinical and genotype data is managed using EXCELL® on a MacIntosh®or Sun Microsystems® computer. Statistical analyses were preformed usingJMP (SAS Institute, Inc. Cary, N.C.). The Wilcoxon/Kruskal-Wallis Tests(rank sums) was used to test whether individuals who were homozygous(Met/Met), heterozygous (Met/Thr), or homozygous (Thr/Thr) at codon 117differ in their serum total IgE. All P-values are two-tailed exceptaffected sib-pair analyses, where a one-tailed test was used becauseonly an increased sharing of alleles was expected.

Having provided this background information, applicant now describespreferred aspects of the invention.

Example 1

Linkage Analysis Between BHR and Murine Chromosome 13

As an aid in dissecting the complex genetic determinants of BHR,applicant has developed murine models that differ in their geneticsusceptibility to various bronchoconstrictor stimuli. Inbred animalmodels using recombinant inbred strains (BXD) can facilitate ongoingstudies in humans to determine the number of genes regulatingsusceptibility to BHR, the magnitude of their affect, and their precisechromosomal location. In particular, localizing in an animal model agene determining susceptibility to a critical risk factor for asthma mayaid in the positional cloning of this gene in humans.

Although the gene(s) predisposing to BHR and atopy had not yet beenidentified prior to this invention, chromosome 5q31-q33 was known to besyntenic with portions of mouse chromosomes 11, 13, and 18. FIG. 2illustrates the syntenic regions containing numerous positionalcandidates that may potentially play a role in airway inflammationassociated with BHR, atopy, and asthma. Specifically, the region ofhuman chromosome 5q31-q33 demonstrating significant evidence for linkagewith BHR is homologous to portions of mouse chromosomes 11, 13, and 18which contain numerous candidate genes.⁸⁴

In particular, IL-9 or a nearby gene have recently been suggested aslikely candidates on the basis of linkage disequilibrium between logserum total IgE levels and a marker in this gene using a randomlyascertained population.⁴³

Despite comparisons with four candidate intervals, evidence for linkagewas found for only one region, designated Aib 1 (atracurium inducedbronchoconstriction 1). FIG. 3 provides the results. Specifically, FIG.3 sets forth the LOD score curve on mouse chromosome 13 foratracurium-induced airway responsiveness in 24 BXD R1 strains which arederived from the hyporesponsive C57BL/6J and the hyperresponsive DBA/2Jprogenitor strains (solid line). The LOD score curve resulting from theselective genotyping of 20 BXD strains is also shown (dashed line). BXDstrains-2, -6, -18, and -32 were not used in the second analysis sincethey were intermediate in phenotype displaying a mean response greaterthan 1 standard deviation below the DBA/2 and above the C57BL/6 meanresponses. The bronchoconstrictor response to atracurium, 20 mg/kg givenintravenously, was assessed by the change in peak inspiratory pressureintegrated over time (4 min), termed the airway pressure time index(APTI). Atracurium-induced APTI was measured in 2–8 animals per R1strain. Marker data were obtained from the RWE data base in the MapManager data analysis program. The genetic distance (cM) between markersis indicated on the abscissa. LOD scores were calculated by theMAPMAKER/QTL linkage program. A QTL was detected in this region andtermed atracurium-induced bronchoconstriction-1 (Aib1).

FIG. 3 indicates that this quantitative trait locus (QTL) is located onthe midportion of murine chromosome 13 and attained this interval amaximum likelihood log of the odds (LOD) of 2.42. Forty-four percent ofthe total variance in atracurium-induced bronchoconstriction wasexplained at Aib1 when all of the markers in the BXD map were analyzed.The LOD for chromosome 13 increased to 2.85 when QTL analyses were runafter excluding the four strains (BXD-2, -6, -18, and -32) that wereintermediate responders to atracurium. The known positional candidatesin the linked region of chromosome 13 include: D1 dopamine receptor(Drd1), fibroblast growth factor receptor 4 (Fgfr4), lymphocyteantigen-28 (Ly28), thiopurine methyltransferase (Tpmt), and IL-9.

Because the applicant was specifically testing for linkage to fourcandidate regions in the mouse, based on previous mapping data in thehuman, the data presented here are highly significant. As stated in theclassic paper by Lander and Botstein,⁶⁷ a false positive rate forlinkage will result if the LOD threshold (T) is chosen so that T=1/2(log10 e)(Z*alpha/n)2 (where n is equal to the number of intervals tested).Typically a minimum LOD of 3.3 is required as evidence of linkage.⁶⁷However, this threshold is based on the assumption that one is searchingthe entire genome. These same authors point out that a LOD of 0.83 issufficient when only one region is examined. In this case, with fourcandidate regions, a P value (alpha) of 0.0125 for each region isrequired to obtain a true P<or =0.05, when one corrects for multipleindependent comparisons. Adopting a 5% error rate that even a singlefalse positive finding will occur, as suggested by Soller and Brody,⁶⁸and solving the equation 1/2(log 10 e) (Z*alpha/n)², yields a LODthreshold of at least 1.36. A maximal LOD of 1.48 was obtained for theIL-9 gene candidate. Restricting the acceptable false positive errorrate to > or =0.1%, increases the LOD threshold to 2.36. Thus, themaximal LOD generated of 2.42 for the candidate interval on chromosome13 (Aib1) is highly significant.

These LOD threshold data provide evidence of a conserved linkage for BHRin humans and mice. BHR in humans links to the region on chromosome 5qcontaining a number of growth factors and cytokines including the IL-9gene and the Aib1 locus maps to the IL-9 region of murine chromosome 13.

Example 2

Identification of an IL-9 Gene Polymorphism

Inventors demonstrated conserved linkage between the mouse and humansfor BHR. These data suggest that variation in the functions of this geneor DNA sequence may be important in regulating bronchial responsivenessin the mouse. Using the methods described above, a unique product of thecorrect size was identified by gel electrophoresis for each of the exonsof human IL-9 after PCR. A single polymorphism was identified by SSCP inexon 5 of the human IL-9 gene. Direct DNA sequence analysis demonstrateda C to T nucleotide substitution at position 3365 (GenBank accessionnumber M30136) of the human IL-9 gene as the cause of the novel SSCPconformer. This DNA sequence change predicts a nonconservativesubstitution of a methionine (hydrophobic) for a threonine (hydrophilic)at amino acid 117 of the IL-9 protein.

Exon 5 codes for this segment of the protein which is within the mosthighly conserved interval of human IL-9 as compared to the mouse IL-9sequence (see FIG. 4).

Individuals were genotyped from various populations to examine thefrequency of these alleles by direct analyses of the nucleotidesubstitution in the coding sequence of human IL-9. Two of 394individuals from a group of asthmatic families were homozygous (Met/Met)at codon 117 (0.5%). There were 91 (23.1%) heterozygous, and 301 (76.4%)homozygous (Thr/Thr) individuals. The true prevalence for this IL-9variant is likely to be significantly higher because the Italianpopulation of families was ascertained through symptomatic patients withasthma. From a separate ethnically diverse population ascertainedrandomly with respect to atopy and asthma, there were 1 of 49individuals homozygous for (Met/Met) at codon 117 (2.0%). There were 11(22.4%) heterozygous, and 37 (75.5%) homozygous (Thr/Thr) individuals.The prevalence of the Met/Thr heterozygotes was 18.9% in a fourthpopulation ascertained randomly with respect to atopy and asthma. Thus,approximately 20% of the population are likely to represent carriers ofthe T allele at position 3,365 as compared to the reported sequence(GenBank accession number M30136). Because it is well known in the artthat the frequency of any allele in the population is p2+2pq+q2, then,approximately 4% of the population is expected to be Met/Met homozygousat codon 117 of IL-9.

Overall, serum total IgE averaged 44.5 I.U. for homozygous individuals(Met/Met), which was significantly different from those who werehomozygous wild type (Thr/Thr) (351.7 I.U.), or heterozygous (Met/Thr)(320.9 I.U.). See FIG. 5. The homozygous protected individuals (Met/Met)failed to demonstrate evidence of atopic allergy except for a singlepositive skin test in one individual. These data indicate that thisnovel DNA polymorphism, when inherited in the homozygous state, isassociated with protection from atopic allergy, including lower serumtotal IgE.

FIG. 6 illustrates the PCR amplification of the IL-9 simple sequencerepeat polymorphism. This marker is compared with genotype for theseindividuals for the restriction fragment length polymorphism produced bythe nucleotide polymorphism at position 3,365 as compared to thereported sequence (GenBank accession number M30136). Two families areshown. The individuals in lanes 1 and 2 are the parents (Thr/Met) ofindividuals in lanes 3 (Met/Thr) and 4 (Met/Met); lanes 5 (Thr/Thr) and6 (Met/Met) are parents for offspring in lanes 7 (Met/Thr) and 8(Met/Thr). The smallest allele for the IL-9 simple sequence repeatpolymorphism (the lowest band in each figure is 248 nucleotides inlength) is in complete linkage disequilibrium with the Met117 allele(nucleotide substitution at position 3,365 as compared to the reportedsequence (GenBank accession number M30136)) in these individuals and inall individuals from populations tested world wide. This was true inboth the Italian and all random ascertained ethnically diverseindividuals studied, and therefore, this marker may be useddiagnostically to detect the presence of the Met117 allele. These dataare most consistent with the hypothesis that this variant is widelydistributed in populations worldwide and arose before many of thesepopulations separated.

Example 3

IL-9 Receptor Expression and Ligand Binding Assay

Purified recombinant Thr IL-9, Met IL-9, and compounds potentiallyresembling Met IL-9 in structure or function are radiolabelled using theBolton and Hunter reagent as described in Bolton A E, and Hunter W M,Biochem J. 133:529–539(1973). This material is labeled to high specificactivity of 2,300 cpm/fmol or greater. Human K562 and MO7e cells aregrown and resuspended at 30° C. in 0.8 ml of Dulbecco's modified Eagle'smedium supplemented with 10% (vol/vol) fetal bovine serum, 50 mM2-mercaptoethanol, 0.55 mM L-arginine, 0.24 mM L-asparagine, and 1.25 mML-glutamine. K562 or MO7e cells are used as is or after transfectionwith the IL-9 receptor gene as described below. Plasmid DNA containingthe full length IL-9 receptor is cloned into pRC/RSV plasmid (InVitrogen, San Diego) and purified by centrifugation through CsCl2.Plasmid DNA (50 micrograms) is added to the cells in 0.4 cm cuvettesjust before electroporation. After a double electric pulse (750V/74ohms/40 microFaradays and 100 V/74 ohms/2100 microFaradays) the cellsare immediately diluted in fresh medium supplemented with IL-9. After 24h the cells are washed and incubated in G418 (2.5 mg/ml, GIBCO) witheither no ligand, or various concentrations of ¹²⁵I-labeled ligand at20° C. for 3 h. An excess of unlabeled ligand is used in parallelexperiments to estimate nonspecific binding. The cells are then washed,filtered, and collected for counting. Specific incorporation iscalculated by Scatchard analysis. Similar competitive assays are runusing ¹²⁵I-labeled Thr117 IL-9 and various amounts of putative coldligands to assess specific binding.

Soluble IL-9 receptor including amino acids 44 to 270 (R&D Systems) wasincubated with different forms of human recombinant IL-9. Varyingamounts of FlagMet117 and FlagThr117 (described in Example 7) wereincubated in PBS at room temperature for 30 minutes with 0.5 microgramsof soluble receptor. EBC buffer (50 mM Tris pH 7.5; 0.1 M NaCl; 0.5%NP40) was added (300 microliters) was added along with 1 microgram ofanti-FLAG monoclonal antibody (IBI) and incubated for 1 hour on ice.Forty microliters of protein A sepharose solution was added to eachsample and mixed for 1 hour at 4° C. Samples were centrifuged for 1minute 11,000 xG and pellets were washed 4 times with 500 microliters ofEBC. Pellets were dissolved in 26 microliters of 2× SDS buffer, boiledfor 4 minutes, and electrophoresed through an 18% SDS polyacrylamidegel. Western blots were performed as described in Example 15 except theblots were probed with an anti-IL-9 receptor antibody (R&D Systems).

FIG. 24 demonstrates the binding of the IL-9 recombinant proteinssoluble IL-9 receptor. Lane 1 is molecular weight markers, lane 2 is theIL-9 FlagMet117 incubated with the receptor, lane 3 is the IL-9FlagThr117 incubated with the receptor. These data demonstrate that bothforms of the recombinant IL-9 protein are bound to the soluble receptor.Moreover, these data along with those of Example 2 (where hetrozygotesdo not differ in serum Ig-E from homozygous Thr117 individuals) areconsistent with the IL-9 Met117 form representing a weak agonist.

Example 4

IL-9 Receptor Expression and Ligand Functional Assay in K562, C8166-45,and MO7e Cells

Recombinant Thr117 IL-9, Met117 IL-9, and compounds potentiallyresembling Met IL-9 in structure or function were purified and preparedfor use in Dulbecco's modified Eagle's medium. K562, C8166-45 or MO7ecells are used as is or after transfection with the IL-9 receptor geneas described in Example 3. After 24 h of deprivation from growth factorsthe cells are incubated without (control) or with variable amounts ofpurified Thr117 IL-9, Met117 IL-9, and compounds potentially resemblingMet117 IL-9 in structure or function. Cellular proliferation is assessedby measuring acid phosphotase activity. Briefly, quadruplicate samplesof MO7e cells are cultured in flat-bottom microtiter plates (150 or 200microliter wells) with or without ligand for 72 to 96 hours at 37° C.Acid phosphatase is measured as suggested by the manufacturer (Clontech,Palo Alto, Calif.). All experiments are repeated at least twice.

Example 5

Cell Isolation and Culture

Human peripheral blood mononuclear cells {PBMC} were isolated fromhealthy donors by density gradient centrifugation using endotoxin testedFicoll-Paque PLUS according to the manufacturer (Pharmacia Biotech, ABUppsala Sweden). PBMC (5×10⁶), mouse spleen cells (5×10⁶), or 5×10⁶ MO7ecells were cultured in 7 ml of RPMI-1640 (Bethesda Research Labs (BRL),Bethesda, Md.) supplemented to a final concentration of 10% with eitherisogenic human serum or heat-inactivated FBS. Cells were cultured for 24hrs at 37° C. either unstimulated, or stimulated with either PMA 5micrograms/ml, PHA 5 micrograms/ml, or PHA 5 micrograms/ml/rhIL2 50U(R&D Systems, Minneapolis, Minn.).

Example 6

RNA Isolations, RT-PCR, Cloning and Sequencing of RT-PCR Products

Total cellular RNA was extracted after 24 hours from cultured PBMC,mouse spleen cells, and MO7e cells using RNA PCR corekit (Perkin-ElmerCorp, Foster City, Calif.) according to the supplier. One microgram ofRNA from each source was denatured for 5 minutes at 65° C. and thenreverse transcribed into cDNA using a 20 microliter reaction mixture(RNA PCR corekit, Perkin-elmer Corp, Foster City, Calif.) containing 50Uof MuMLV Reverse Transcriptase, 1U/microliter RNAse Inhibitor, 2.5 mMoligo d(T)16 primer, 1 mM each of DATP, dCTP, dGTP, dTTP, 50 mM KCl, 10mM Tris-HCL, pH 7.0, 25 mM MgCl2. The reaction mixture was pipetted intothermocycler tubes, placed in a PCR thermal cycler and subjected to 1cycle (15 minutes at 42° C., 5 minutes at 99° C., and 5 minutes at 4°C.). A mock reverse transcription reaction was used as a negativecontrol.

Then this mixture was added to a second tube containing 2 mM MgCl2, 50mM KCl, 10 mM Tris-HCl, pH 7.0, 65.5 microliters of DI water, 2.5UAmplitaq DNA polymerase, and 1 microliter (20 micromolar) each ofoligonucleotides representing human cDNA IL-9 exon 1 (forward) and exon5 (reverse), for a final volume of 100 microliters. The reaction mixturewas subjected to the following PCR conditions: 120 seconds at 98° C.,then 30 cycles at: 30 seconds at 94° C.; 40 seconds at 55° C.; 40seconds at 72° C. Finally, the reaction mixture was cycled one time for15 minutes at 72° C. for extension.

PCR products representing hIL-9 cDNA were subjected to gelelectrophoresis through 1.5% agarose gels and visualized using ethidiumbromide staining. Products of a mock reverse transcriptase reaction, inwhich H₂O was substituted for RNA, and used as negative controlamplification in all experiments.

The PCR oligonucleotide primer pairs used in these experiments toamplify cDNA include: human interleukin 9 (hIL-9) exon 1 forward 5′-TCTCGA GCA GGG GTG TCC AAC CTT GGC G-3′ (SEQ ID NO: 1) and exon 5 reverse5′ GCA GCT GGG ATA AAT AAT ATT TCA TCT TCA T-3′ (SEQ ID NO: 2); mouseinterleukin 9 (mIL-9) exon 1 forward 5′-TCT CGA GCA GAG ATG CAG CAC CACATG GGG C-3′ (SEQ ID NO: 3) and mouse exon 5 reverse 5′-GCA GCT GGT AACAGT TAT GGA GGG GAG GTT T-3′ (SEQ ID NO: 4); XhoI and PvuII restrictionenzyme recognition sequences are underlined in the human and mouse IL-9primers. PCR products were subcloned into the TA Cloning vector(Invitrogen, San Diego, Calif.). Amplification of the mouse cDNA gave a438 bp product and amplification of the human cDNA gave a 410 bpproduct.

Complementary DNAs for human IL-9 and murine mIL-9 were generated andamplified by RT-PCR using IL-9 exon 1 and 5 specific primers containingdigestion sites for XhoI and PvuII restriction endonucleases.Amplification products for hIL-9 and mIL-9 were isolated from 2.5agarose gels using silica (Sambrook et al., (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press) (incorporatedherein by reference in its entirety). After recovery, the cDNA productswere ligated into the TA Cloning vector (Invitrogen Corp., San Diego,Calif.) and then used to transform INV-alpha-F′ competent cells,according to the manufacturer's instructions. Plasmids containing hIL-9and mIL-9 cDNA inserts were isolated by conventional techniques(Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual ColdSpring Harbor Laboratory Press). After amplification the DNA sequenceincluding and surrounding each insert was analyzed for PCR-induced orcloning-induced errors.

hIL-9 and mIL-9 cDNA inserts were sequenced by the dideoxy-mediatedchain termination method (Sanger et al., (1977) Proc. Natl. Acad. Sci.USA 74, 5463–5467), using the M13 (−20) forward primer (5′-GTA AAA CGACGG CCA GT-3′) (SEQ ID NO: 18) and Sequenase™ (USB), and analyzed by gelelectrophoresis (Sambrook et al., (1989) Molecular cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press). hIL-9 and mIL-9 cDNAinserts without cloning and/or Taq polymerase-induced sequence errors(see translated cDNA sequences FIGS. 7 and 8) were subcloned intoexpression vectors (see FIGS. 9–12) or used to create missense mutationsand deletion mutants.

Example 7

Cloning and Expression of IL-9 Constructs in Vitro General CloningMethods for Constructs

hIL-9 was subcloned into procaryotic expression vectors. The TA2AAF1 metand thr vectors were digested by EcoRI and the 0.420 kB fragment(containing an XhoI site at the 5′ end of the hIL9 cDNA) was cloned intothe EcoRI site contained with the polylinker of pBluescript (PBS)(Stratagene). Clones in the sense orientation to the T3 promoter werethen digested with XhoI (the fragment contained a 5′ XhoI site from theIL-9 cDNA insert from TA vectors and a 3′ XhoI site from the PBSpolylinker) and inserts were subcloned into the XhoI sites of theprocaryotic expression vectors PGEX and PFLAG.

Cloning and Expression of IL-9 Constructs in the pGEX-4T-1 Glutathiones-transferase Gene Fusion Vector

For the expression, purification, and detection of IL-9 protein, IL-9cDNA inserts were subcloned into the XhoI site within the multiplecloning cassette of the 4.9 Kb pGEX-4T-1 glutathione s-transferase genefusion vector (Pharmacia Biotech, Piscataway, N.J.) by standardtechniques. Briefly, TA clones containing intact IL-9 cDNA sequences,and the pGEX-4T-1 vector were digested for one hour at 37° C. using XhoIand PvuII restriction endonuclease in the presence of 1× React 2 buffer(New England Biolabs, Beverly, Mass.) (total volume 50 microliters).Products were electrophoresed in a 1.5% preparative agarose gel with 10micrograms/ml ethidium bromide. The appropriate sized DNA band wasexcised, the agarose was melted at 45° C. for 10 minutes in 3 volumes ofNaI stock solution. A silica matrix solution in DI H₂O (Geneclean II,LaJolla, Calif.) was added to the solution at 5 microliters per 5micrograms of DNA and 1 microliter per 0.5 micrograms of DNA above 5micrograms. The slurry was incubated at 4° C. and occasionally shakenduring 30 minutes. The slurry was then pelleted via microcentrifugation,washed 3 times in low-salt buffer and resuspended in 10 microliters ofDI H₂O to elute the DNA from the silica. A final microcentrifugationprovided the 10 microliter solution containing purified DNA.

Products were resuspended in 50 microliters of DI H₂O and precipitatedby the addition of 2 volumes of ethanol and 1/10 volume 3M sodiumacetate. Samples were centrifuged at RT at 14,000 rpm for 10 minutes,air dried under negative pressure and resuspended in an appropriatevolume of DI H₂O. Ligations and transformations of DH5a bacteria(GIBCO/BRL, Gaithersburg, Md.) with mIL-9 and hIL-9 cDNA inserts in thepGEX-4T-1 vector were performed using standard techniques.

To confirm that the hIL-9 cDNA inserts contained in the pGEX-4T-1 vectorwere of the correct nucleotide sequence, plasmids containing candidateIL-9 cDNA were sequenced via the dideoxy-mediated chain terminationmethod using the aforementioned mIL-9 and hIL-9 cDNA-specificoligonucleotides (exon 1 forward, exon 5 reverse primers).

Recombinant fusion, proteins were obtained from large scale cultures.The overnight culture of transformed E. Coli (50 ml) was inoculated intofresh LB/amp broth. The culture was incubated for 4 hr at 37° C. withvigorous shaking, isopropyl-beta-D-thiogalactopyranoside was then addedto a final concentration of 1 mM, and the culture was incubated for anadditional 1.5 hours. The cells are harvested by centrifugation at 500×gat 4° C. and recombinant variants were purified by making use ofaffinity chromatography on glutathione-sepharose 48 column (Pharmacia)for GST-fusion proteins. Some variants were expressed as inclusionbodies and were purified from insoluble inclusion bodies by theprocedure described by Marston (1987) The purification of eukaryoticpolypeptides expressed in E. coli in Clover D. M. ed. DNA cloning: Apractical approach, IRL Press, Oxford, 59–88). Briefly, the cells werelysed with lysozyme followed by treatment with deoxycholic acid.Contaminating nucleic acids were removed by treatment finallysolubilized in lysis buffer (50 mM Tris-Cl, pH 8.0, 1 mM EDTA, 100 mMNaCl) containing 8 M urea. The solubilized components from the inclusionbodies were dialyzed stepwise against decreasing concentrations of urea(starting with 8, 6, 4 and 2 M of urea) in lysis buffer to allow forrefolding of the denatured protein. Finally, the sample was dialyzedagainst 2 M urea and 2.5% beta-mercaptoethanol (beta-ME) and centrifugedat 10,000 g for 15 minutes. The fusion protein was finally dialyzedagainst 0.01 M Tris-Cl, pH 8.0. Fusion proteins expressed in PGEX-4Tvector were cleaved with 100 U of Thrombin for 6 hours at 37° C. andrecovered in flow through fractions after chromatography onglutathione-Sepharose 4B column. Final purification was achieved bychromatography on Sephadex G-100 column (100×1.5 cm), packed andequiliberated with 0.05 M ammonium bicarbonate buffer.

Cloning and Expression of IL-9 Constructs in the pFLAG-1™ExpressionVector

For the expression, purification and detection of human IL-9 protein,IL-9 cDNA inserts were subcloned into the Xho2 site of the multiplecloning site (XhoI) of the 5.37 Kb Flag vector. FLAG technology iscentered on the fusion of a low molecular weight (1 kD), hydrophilic,FLAG marker peptide to the N-Terminus of a recombinant protein expressedby the pFLAG-1™ Expression Vector (obtained from IBI Kodak). Eachbacterial colony was grown in LB broth containing 50 microgramsampicillin per ml until the optical density at 590 nm reached 0.6. IPTGwas then added to a final concentration of 1 mM, and the culturesincubated for an additional 1 hr to induce protein synthesis. The cellswere harvested by centrifugation, and the cell pellet was boiled in 50microliters of Laemmli buffer (Laemmli, (1970)) for 10 minutes andelectrophoresed on 10% polyacrylamide gels. The Anti-FLAG™ M1 monoclonalantibody was used for specific and efficient detection of the FLAGfusion protein on western (see FIG. 13) slot or dot blots throughout itsexpression, affinity purification, and FLAG marker removal. The FLAGfusion protein was rapidly purified under mild, non-denaturingconditions in a single step by affinity chromatography with the murineAnti-FLAG™ IgG M1 monoclonal antibody covalently attached to agarose.Following affinity purification the fusion protein may be used afterremoval from the affinity column or the authentic protein may berecovered in biologically active form by specific and efficientproteolytic removal of the FLAG peptide with enterokinase. Finalpurification was achieved by chromatography on Sephadex G-100 column(100×1.5 cm), packed and equiliberated with 0.05 M ammonium bicarbonatebuffer. The promoters described above in this example may also be usedwith FLAG technology.

SDS-PAGE and Immunoblot Analysis

SDS-PAGE was performed by the method of Laemmli (Laemmli U.K. (1970)Nature 227, 680–685)(incorporated herein by reference in its entirety)by using a 12.5% polyacrylamide gel in a mini-gel system (SE 280vertical gel unit, Hoefer). For immunoblot analysis, the proteinsseparated by SDS-PAGE were transferred to nitrocellulose membranes byusing the TE 22 Mighty small transfer unit (Hoefer) in 25 mMTris-glycine buffer, pH 8.3, containing 15% methanol (Towbin et al.,(1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350–4354). The unoccupiedbinding sites on the membrane were blocked by incubating for 1 h with 20mM Tris-HCl buffer, pH 8.0, containing 2% bovine serum albumin. Themembranes were then incubated with 1:200 dilution of antibodiesovernight at 4° C. The membranes were washed and treated with 1:2000diluted goat anti-rabbit IgG conjugated with either peroxidase oralkaline phosphotase for 1 h. After washing, the bound antibodies werevisualized by addition of the supersubstrate chemiluminescent reagent(Pierce) or the 4-chloro-1-naphthol color developing reagent. Thereaction was stopped by immersing the membranes in distilled water. FIG.13 demonstrates that the purified recombinant human FLAG IL-9 fusionproteins (Met117 and Thr117) are the correct size and in the correctreading frame because they are recognized by the Anti-FLAG™ M1monoclonal antibody.

Analytical Methods

The molecular mass of the purified proteins was confirmed by matrixassisted laser desorption mass spectrometry using Perceptive BiosystemsVoyager Biospectrometry workstation. Amino acid analyses were performedafter hydrolysis of the sample in 6N HCl at 110-C for 24 h in evacuatedsealed glass bulbs.

Automated Edman Degradation

The partial amino acid sequence of the purified proteins is determinedby automated step-wise sequencing on an Applied Biosystems model 477Agas-phase sequencer with an on-line model 20A PTH analyzer.

Example 8

Deletion of Exon 2 and/or Exon 3: Mutagenesis and Sequencing ofConstructs

Human exon 2 and exon 3 deletions are created using ExSite PCR-basedsite-directed mutagenesis kit as suggested by the manufacturer(Stratagene, La Jolla, Calif.). The PCR primers are as follows: h9CD1Uforward 5′-GTG ACC AGT TGT CTC TGT TTG-3′ (SEQ ID NO: 5); h9CD1L reverse5′-CTG CAT CTT GTT GAT GAG GAA-3′ (SEQ ID NO: 6); h9CD2U forward 5′-GACAAC TGC ACC AGA CCA TGC-3′ (SEQ ID NO: 7); h9CD2L reverse 5′-ATT AGC ACTGCA GTG GCA CTT-3′ (SEQ ID NO: 8). Exon 2 deletions are created by usingthe primer pair h9CD1L forward and h9CD1L reverse. Exon 3 deletions arecreated by using h9CD2U forward and h9CD2L reverse. Deletions thatincluded exon 2 and exon 3 use the primer pair h9CD2U forward h9CD1Lreverse.

Mouse exon 2 and exon 3 deletions are created using ExSite PCR-basedsite-directed mutagenesis kit as suggested by the manufacturer(Stratagene, La Jolla, Calif.). The PCR primers are as follows: m9CD1Uforward 5′-GTG ACC AGC TGC TTG TGT CTC-3′ (SEQ ID NO: 9); m9CD1L reverse51-CTT CAG ATT TTC AAT AAG GTA-3′ (SEQ ID NO: 10); m9CD2U forward 5′-GATGAT TGT ACC ACA CCG TGC-3′ (SEQ ID NO: 11); m9CD2L reverse 5′-GTT GCCGCT GCA GCT ACA TTT-3′ (SEQ ID NO: 12). Exon 2 deletions are created byusing the primer pair m9CD1U forward and m9CD1L reverse. Exon 3deletions are created by using m9CD2U forward and m9CD2L reverse.Deletions that included exon 2 and exon 3 use the primer pair m9CD2Uforward m9CD1L reverse.

Mutagenized constructs of the hIL-9 and mIL-9 cDNA inserts are sequencedby the dideoxy-mediated chain termination method (Sanger et al. (1977)Proc. Natl. Acad. Sci. USA 74, 5463–5467) (incorporated herein byreference in its entirety), using the M13 (−20) forward primer(5′-GTAAAACGACGGCCAGT-3′) (SEQ ID NO: 18) and Sequenase™ (USB), withanalysis by gel electrophoresis (Sambrook et al. (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press).Mutants that lack exon 2, exon 3, or both exon 2 and exon 3, and arewithout Taq polymerase-induced sequence errors can be used to createexpression vectors.

Example 9

Cell Lines, Cellular Proliferation Assays and Inhibition of IL-9Activity

Cell lines were used to assess the function of peptides, aminosterols,tyrophostins, rhIL-9, rmIL-9, and recombinant mutant forms of theseproteins as well as all other compounds that block IL-9 function. Aproliferative response was measured and compared to each of the othercytokines, variant or mutant forms of Il-9, or IL-9 antagonists. Inaddition, compounds were tested for their ability to antagonize thebaseline proliferative response. Once a baseline proliferative responsewas established for a cytokine a statistically significant loss ofresponse in assays repeated three times in triplicate was consideredevidence for antagonism. A true antagonistic response was differentiatedfrom cellular toxicity by direct observation, trypan blue staining (atechnique well known to one of normal skill in the art), and loss ofacid phosphatase activity. Specificity was assessed for the antagonistby evaluating whether the activity was substantially expressed againstother proliferative agents such as steel factor, interleukin 3, orinterleukin 4.

The MO7e line is a human megakaryoblastic cell line, cultured in RPMI1640 (GIBCO/BRL, Gaithersburg, Md.), 20% Fetal Bovine Serum (Hyclone)and 10 ng/ml IL-3 (R&D Systems, Minneapolis, Minn.). The MJ line is acytokine independent human lymphoblastoid cell line grown in RPMI 1640(GIBCO/BRL) K562 is a human erthroleukemia cell line, cultured in RPMI1640 (GIBCO/BRL) and 10% fetal bovine serum (Hyclone). C8166-45 is aIL-9 receptor bearing line, cultured in RPMI 1640 GIBCO/BRL) and 10%Fetal bovine serum (Hyclone). All the cell lines respond to cytokinesincluding IL-9. The cell lines are fed and reseeded at 2×10⁵ cells/mlevery 72 hours.

The cells were centrifuged for 10 minutes at 2000 rpm and resuspended inRPMI 1640 with 0.5% Bovine Serum Albumin (GIBCO/BRL, Gaithersburg, Md.)and insulin-transferrin-selenium (ITS) cofactors (GIBCO/BRL,Gaithersburg, Md.). Cells were counted using a hemocytometer and dilutedto a concentration of 1×10⁵ cells/ml and plated in a 96-well microtiterplate. Each well contained 0.15 or 0.2 ml giving a final concentrationof 2×10⁴ cells per well.

MO7e cells were stimulated with 50 ng/ml Stem Cell Factor (SCF) (R&DSystems, Minneapolis, Minn.) alone, 50 ng/ml SCF plus 50 ng/ml IL-3 (R&DSystems, Minneapolis, Minn.), or 50 ng/ml SCF plus 50 ng/ml IL-9. Acontrol was included which contains cells and basal media only. Serialdilutions of test compounds (i.e., recombinant IL-9 proteins, peptides,small molecules) were added to each test condition in triplicate. The MJcell line was used as an independent control for nonspecificcytotoxicity. Cultures were incubated for 72–96 hours at 37° C. in 5%CO₂.

Cell proliferation was assayed using the Abacus Cell Proliferation Kit(Clontech, Palo Alto, Calif.) which determines the amount ofintracellular acid phosphatase present as an indication of cell number.The substrate p-nitrophenyl phosphate (pNPP) was converted by acidphosphatase to p-nitrophenol which was measured as an indicator ofenzyme concentration. pNPP was added to each well and incubated at 37°C. for one hour. 1N sodium hydroxide was then added to stop theenzymatic reaction, and the amount of p-nitrophenol was quantified usinga Dynatech 2000 plate reader (Dynatech Laboratories, Chantilly, Va.) at410 nm wavelength. Standard curves that compare cell number with opticalabsorbance were used to determine the linear range of the assay. Assayresults were only used when absorbance measurements are within thelinear range of the assay.

FIG. 14 illustrates the amino acid sequence of three peptide antagonistsof IL-9 function. Each peptide was incubated with MO7e cells andinhibition of cellular growth induced by IL-9 was determined bycomparison with control conditions (no peptide)(see FIGS. 15–17). Therewas no evidence for cytotoxicity with any of the peptides. PeptidesKP-16 and KP-20 are predicted to lie within two anti-parallelalpha-helicies and define a critical IL-9 receptor binding domain forthe IL-9 ligand. The protein polymorphism at codon 117 lies within KP-23and KP-24 which also exhibited antagonistic properties, furtherdemonstrating the importance of this region surrounding the site ofgenetic variation.

FIG. 18 illustrates the effect of tyrophostins (obtained fromCalbiochem) on the IL-9 dependent growth of MO7e cell in vitro. Eachtyrophostin was incubated with MO7e cells and inhibition of cellulargrowth induced by IL-9 was determined by comparison with controlconditions (no treatment). There was no evidence for cytotoxicity withany of the treatments. Tyrophostins B46 and B56 provided the greatestinhibition suggesting a common structure activity relationship.

FIG. 19 illustrates the effect of aminosterols isolated from the sharkliver as set forth in U.S. Pat. Nos. 5,637,691; 5,733,899; 5,721,226and/or 5,840,740 (incorporated herein by reference) on the IL-9dependent growth of MO7e cell in vitro. Each aminosterol was incubatedwith MO7e cells at 20 micrograms/ml of the culture media and inhibitionof cellular growth induced by IL-9 was determined by comparison withcontrol conditions (no treatment). There was no evidence forcytotoxicity with any of the treatments. Aminosterols 3 and 6consistently provided the greatest inhibition of growth.

Example 10

Assay for Proliferation of IgE Secreting Cells

B cell lines can be used to assess the function of rIL-9 and recombinantmutant forms of these proteins as well as other IL-9 antagonists. Theproliferation of IgE secreting cells is measured for rIL-9 and comparedto other cytokines or variant forms of rIL-9. In addition, compounds aretested for their ability to antagonize the baseline proliferativeresponse of IgE secreting cells to rIL-9. Once a baseline IgE responseis established for a cytokine, a statistically significant (P<0.05) lossof response in assays repeated three times in triplicate is consideredevidence for antagonism. A true antagonistic response is differentiatedfrom cellular toxicity by trypan blue staining (a technique well knownto one of normal skill in the art).

Cell Preparation and Cultures

Peripheral blood lymphocytes (PBL) are isolated from heparinized bloodof healthy donors or by mincing the spleens of mice. Mononuclear cellsare separated by centrifugation on Ficoll/Hypaque (Pharmacia, Uppsala,Sweden) gradients. Semi-purified human B lymphocytes are obtained byresetting with neuraminidase (Behring, Marburg, FRG)—treated sheep redblood cells and plastic adherence for 1 hour at 37° C. B cells are alsopurified using paramagnetic separation with anti-CD20 coated magneticbeads (DYNAL) according to the manufacturer's recommendations.

The relative proportion of B cells, T cells and monocytes is determinedby flow cytometry using monoclonal antibodies specific for CD23, CD3 andCD14, respectively (Becton Dickinson, Mountain View, Calif.). Briefly,10⁶ cells/ml are incubated with a 1:1000 dilution of phycoerythrinconjugated anti-CD23 and fluoresceinconjugated anti-CD3 and anti-CD14antibodies for 30 minutes at 4° C. After 3 spin washes with sterile PBSand 1% bovine serum albumin (Sigma) fluorescence is measured with acytofluorograph (FACSTAR Plus, Becton Dickinson, Grenoble, France).Typically, there are 45% CD20+, 35% CD3+ and 10% CD14+ cells in a countof 5000 cells per sample.

Cells are cultured at a density of 2×10⁶ cells/ml RPM1 1640 supplementedwith 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 100U/ml penicillin, 100 micrograms/ml streptomycin and 20 mM HEPES(RPM1-FCS) at 37° C. under a 5% CO₂/95% air humidified atmosphere.Cultures are incubated with increasing concentrations of IL-4, rhIL-9,rmIL-9, or recombinant mutant forms of these proteins, alone, or incombination. Competition experiments are run with mixtures of one ormore of these recombinant molecules or other IL-9 antagonists. Thecultures are maintained for 9–13 days.

Frequency of IgE-Secreting B Cells

The frequency of IgE-secreting human B cells in response to human ormurine IL-9 is determined using an ELISA-spot assay (Dugas et al.,(1993) Eur J Immunol 23, 1687–1692; Renz et al., (1990) J. Immunology.145, 3641–3644). Nitrocellulose flat-bottomed 96-well plates are coatedovernight at 4° C. with purified goat antihuman IgE mAb diluted in 0.1 MNaHCO₃ buffer (2.5 micrograms/ml). After one PBS-Tween 20 wash, platesare incubated for 1 hour with RPMI-FCS to saturate nonspecific bindingsites. B cells obtained after 9–13 days of culture are collected, washedthrice and resuspended at 10⁵ cells/ml RPMI-FCS, then transferred ontothe anti-IgE-coated plates followed by an 18 hour incubation at 37° C. Aperoxidase-conjugated mouse antihuman IgE mab at various dilutions isadded for 2 hours at 37° C. after washing. Spots are visualized afteraddition of diamino-benzidine diluted in 0.1 M Tris-HCl containing0.030% H₂O₂. After 24 hours spots are counted with an invertedmicroscope at 25× magnification. Data are expressed as the number ofIgE-secreting cells per 10⁶ cells.

Example 11

ELISA for IgE Secreted by Cells Co-stimulated with IL-9

Cells are isolated, prepared and stimulated as described above inExample 10. Flat bottom microtiter plates (Nunc) are coated with rabbitanti-human IgE (1:2000, final dilution; Serotec, Oxford, GB), in 200microliters of 10 mM bicarbonate buffer (pH 9.6). After overnightincubation at 4° C., the plates are washed four times withphosphate-buffered saline (PBS) containing 0.05% Tween (PBS-Tween;Merck, Hohenbrunn, FRG) and are incubated for 1 h at room temperaturewith RPMI-FCS to saturate nonspecific protein-binding sites. Afterwashing, 200 microliter serial dilutions of human IgE (Eurobio, LesUlis, France), standards in PBS-Tween are added to the respective platesto establish calibration curves. Dilutions of culture supernatants to betested are then added and, after 2 h at room temperature, the plates arewashed and 200 microliters of diluted specific alkalinephosphatase-conjugated anti-IgE (1:250; Serotec), anti-IgG or anti-IgM(Behring) is added in the appropriate plates. After 2 h at roomtemperature, the plates are washed and 200 microliters (0.5 mg/ml)p-nitrophenylphosphate (Sigma) in citrate buffer is added. Plates areincubated at 37° C., and absorbance (A) is measured at 405 nm using anautoreader (Dynatech Laboratories Inc, Chantilly, Va.). The thresholdsensitivities of the assays are 100 pg/ml for IgE, 1 ng/ml for IgG, and2 ng/ml for IgM and the variation between duplicate determinations ofsamples typically does not exceed 10%.

Example 12

The Role of IL-9 in Murine Models of Asthma: The Airway Response ofUnsensitized Animals Animals

Certified virus-free male mice ranging in age from 5 to 6 weeks wereobtained from the Jackson Laboratory (Bar Harbor, Me.). Animals werehoused in high-efficiency particulate filtered air (HEPA) laminar flowhoods in a virus and antigen free facility and allowed free access topelleted rodent chow and water for 3 to 7 days prior to experimentalmanipulation. The animal facilities were maintained at 22° C. and thelight:dark cycle was automatically controlled (10:14 h light:dark). Maleand female DBA/2 (D2), C57BL/6 (B6), and (B6D2)F1 (F1) mice 5 to 6 weeksof age were purchased from the Jackson Laboratory, Bar Harbor, Me., orthe National Cancer Institute, Frederick, Md. BXD mice were purchasedfrom the Jackson Laboratory, Bar Harbor, Me. Food and water were presentad libitum.

Phenotyping and Efficacy of Pretreatment

To determine the bronchoconstrictor response, respiratory systempressure was measured at the trachea and recorded before and duringexposure to the drug. Mice were anesthetized and instrumented aspreviously described. (Levitt et al., (1988), FASEB J. 2, 2605–2608(1988); Levitt et al., (1989), J. Appl. Physiol. 67, 1125–1132;Kleeberger et al., (1990) Am. J. Physiol. 258, L313–320; Levitt (1991)Pharmacogenetics 1, 94–97; Levitt et al., (1995) Am. J. Respir. Crit.Care Med. 151, 1537–1542; Ewart et al., (1995) J. Appl. Phys. In press.Airway responsiveness was measured to 5-hydroxytryptamine (5HT) (Sigma).Additional broncoconstrictors that can be used are acetylcholine (Sigma)and atracurium (Glaxo Welcome). A simple and repeatable measure of thechange in Ppi following bronchoconstrictor challenge was used and whichhas been termed the Airway Pressure Time Index (APTI) (Levitt et al.,(1988) FASEB J. 2, 2605–2608; Levitt et al., (1989) J. Appl. Physiol.67, 1125–1132. The APTI was assessed by the change in peak inspiratorypressure (Ppi) integrated from the time of injection till the peakpressure returned to baseline or plateaued. The APTI was comparable toairway resistance (Rrs), however, the APTI includes an additionalcomponent related to the recovery from bronchoconstriction.

The strain distribution of bronchial responsiveness was identified inmultiple inbred mouse strains in previous studies (Levitt et al., (1988)FASEB J 2, 2605–2608; Levitt et al., (1989) J. Appl. Physiol. 67,1125–1132). The Rrs and/or APTI was determined in A/J, C3H/HeJ, DBA/2J,C57BL/6J mice.

Prior to sacrifice whole blood was collected for serum IgE measurementsby needle puncture of the inferior vena cava in completely anesthetizedanimals. The samples were spun to separate cells and serum was collectedand used to measure total IgE levels. Samples not measured immediatelywere frozen at negative 20° C. Bronchoalveolar lavage (BAL) and cellularanalyses was preformed as described elsewhere (Kleeberger et al.,(1990)).

All IgE serum samples were measured using an ELISA antibody-sandwichassay. Microtiter plates (Corning #2585096, Corning, N.Y.) were coated,50 microliters per well, with rat anti-mouse IgE antibody (SouthernBiotechnology #1130-01, Birmingham, Ala.) at a concentration of 2.5micrograms/ml in coating buffer of sodium carbonate-sodium bicarbonatewith sodium azide (Sigma #S-7795, #S-6014 and #S-8032, St Louis, Mo.).Plates were covered with plastic wrap and incubated at 4° C. for 16hours. The plates were washed three times with a wash buffer of 0.05%Tween-20 (Sigma #P-7949) in phosphate-buffered saline (BioFluids #313,Rockville, Md.), incubating for five minutes for each wash. Blocking ofnonspecific binding sites was accomplished by adding 200 microliters perwell 5% bovine serum albumin (Sigma #A-7888) in PBS, covering withplastic wrap and incubating for 2 hours at 37° C. After washing threetimes with wash buffer, duplicate 50 microliter test samples were addedto the wells.

Test samples were assayed after being diluted 1:10, 1:50, and 1:100 with5% BSA in wash buffer. In addition to the test samples a set of IgEstandards (PharMingen #03121D, San Diego, Calif.) at concentrations from0.8 ng/ml to 200 ng/ml in 5% BSA in wash buffer were assayed to generatea standard curve. A blank of no sample or standard was used to zero theplate reader (background). After adding samples and standards, the platewas covered with plastic wrap and incubated for 2 hours at roomtemperature. After washing three times with wash buffer, 50 microlitersof second antibody rat anti-mouse IgE-horseradish peroxidase conjugate(PharMingen #02137E) was added at a concentration of 250 ng/ml in 5% BSAin wash buffer. The plate was covered with plastic wrap and incubated 2hours at room temperature. After washing three times with wash buffer,100 microliters of the substrate 0.5 mg/ml O-phenylaminediamine (Sigma#P-1526) in 0.1 M citrate buffer (Sigma #C-8532) was added to everywell. After 5–10 minutes the reaction was stopped with 50 microliters of12.5% H₂SO₄ (VWR #3370-4, Bridgeport, N.J.) and absorbance was measuredat 490 nm on a Dynatech MR-5000 plate reader (Chantilly, Va.). Astandard curve was constructed from the standard IgE concentrations withantigen concentration on the x axis (log scale) and absorbance on they-axis (linear scale). The concentration of IgE in the samples wasinterpolated from the standard curve.

Example 13

The Role of IL-9 in Murine Models of Asthma: The Airway Response ofSensitized Animals

Animals, Phenotyping, and Optimization of Antigen Sensitization

Animals and handling were essentially as described in Example 12.Sensitization with turkey egg albumin (OVA) and aerosol challenge wascarried out to assess the effect on BHR, BAL, and serum IgE. OVA wasinjected I.P. (25 micrograms) day 0 prior to OVA or salineaerosolization. Mice were challenged with OVA or saline aerosolizationwhich was given once daily for 5 to 7 days starting on either day 13 or14. Phenotypic measurements of serum IgE, BAL, and BHR was carried outon day 21. The effect of a 7 day OVA aerosol exposure onbronchoconstrictor challenge with 5-HT and acetylcholine were evaluatedalong with serum total IgE, BAL total cell counts and differential cellcounts, and bronchial responsiveness. The effect of antibody (Ab) orsaline pretreatment on saline aerosol or OVA aerosol induced lunginflammation was examined by measuring BHR, BAL, and serum IgE. Ab wereadministered I.P. 2–3 days prior to aerosolization of saline or OVA.

Lung histology was carried out after the lungs are removed during deepanesthesia. Since prior instrumentation may introduce artifact, separateanimals were used for these studies. Thus, a small group of animals wastreated in parallel exactly the same as the cohort undergoing variouspretreatments except these animals were not used for other tests asidefrom bronchial responsiveness testing. After bronchial responsivenesstesting, the lungs were removed and submersed in liquid nitrogen.Cryosectioning and histologic examination were carried out in a routinefashion.

Polyclonal neutralizing antibodies for murine IL-9 were purchased from R& D systems, Minneapolis, Minn. and blocking antibodies for murine IL-9receptor were produced for Magainin Pharmaceuticals Inc. by LampineBiological Labatories, Ottsville, Pa. using peptide conjugates producedat Magainin. The polyclonal antisera were prepared in rabbits againstpeptide sequences from the murine IL-9 receptor. The peptides used toproduce the antisera were: GGQKAGAFTC (residues 1–10)(SEQ ID NO: 19);LSNSIYRIDCHWSAPELGQESR (residues 11–32)(SEQ ID NO: 20); andCESYEDKTEGEYYKSHWSEWS (residues 184–203 with a Cys residue added to theN-terminus for coupling the peptide to the carrier protein)(SEQ ID NO:21). The antisera were generated using techniques described in Protocolsin Immunology, Chapter 9, John Wiley & Sons. Briefly, the peptides werecoupled to the carrier protein, Keyhole Limpet hemocyanin (Sigma)through the side chain of the Cys residue using the bifunctionalcross-linking agent MBS (Pierce). Peptide conjugates were used toimmunize rabbits with appropriate adjuvents and useful antisera wasobtained after several booster injections of the peptide conjugate. Theantibodies were used therapeutically to down regulate the functions ofIL-9 and assess the importance of this pathway to baseline lungresponsiveness, serum IgE, and BAL in the unsensitized mouse. After Abpretreatment on baseline BHR, BAL, and serum IgE levels relative tocontrols was determined. In additional experiments, recombinant humanand murine IL-9 were administered I.P. 1 day before and daily duringantigen sensitization (days 13–18). The animals were then phenotyped asdescribed.

The phenotypic response of a representative animal treated with salineI.P. on day zero and challenged on days 14–20 with saline (as describedin Example 12) is shown in FIG. 20A panel 1 (top). Baseline (control)serum total IgE was 9.2 ng/ml. Bronchoalveolar lavage (BAL) total cellcounts showed 182,500 cells per milliliter of BAL. These animals did notdemonstrate bronchial hyperresponsiveness when compared to historicalcontrols (Levitt et al., (1989) J. Appl. Physiol. 67, 1125–1132).

FIG. 20A panel 2 (top middle) shows a representative animal from a grouppresensitized with OVA I.P on day zero and challenged with saline ondays 14–20. These animals did not differ in their response tobronchoconstrictor, serum IgE, or BAL cell counts from the unsensitizedmice (FIG. 7 top panel).

FIG. 20B panel 3 (bottom middle) shows a representative animal fromthose presensitized with OVA I.P. on day zero and challenged withantigen (OVA) on days 14–20. These animals developed bronchialhyperresponsiveness (approximately two to three-fold over controls),elevated serum IgE (nearly one thousand-fold over controls), andincreased numbers of inflammatory cells in the airway as demonstrated byelevated BAL cell counts (approximately thirty-fold) as compared tocontrols (FIGS. 20A and 20B top 2 panels). Most of the cells recruitedto the airway as a result of this antigen challenge were eosinophils.

FIG. 20B panel 4 (bottom) shows a representative animal from thosepresensitized with OVA I.P on day zero, pretreated with polyclonalneutralizing antibodies for murine IL-9 (approximately 200micrograms/mouse I.P. in 0.5 ml of PBS), and challenged with antigen(OVA) on days 14–20. These animals were protected from the response toantigen. They did not differ significantly in their bronchialresponsiveness, serum IgE, or BAL cell counts from controls (FIGS. 20Aand 20B top 2 panels).

FIG. 21 illustrates the effect of antigen challenge to OVA (as describedabove) with and without pretreatment with polyclonal neutralizingantibodies to murine IL-9 I.P. three days prior in representativeanimals. The left figure (A1-2-1B) is a histologic section from thelungs of control animals (sensitized to OVA but exposed only to a salineaerosol challenge). The middle figure (A1-3-5) is a histologic sectionfrom the lungs of animals sensitized to OVA and exposed to an OVAaerosol challenge. The right figure (A1-4-5) is a histologic sectionfrom the lungs of animals sensitized to OVA and exposed to an OVAaerosol challenge who were pretreated three days prior with polyclonalneutralizing antibodies to murine IL-9. Pretreatment with neutralizingantibody produced histological confirmation of complete protection fromantigen challenge.

FIG. 22 panel 1 (top) shows a representative animal from micepresensitized with OVA I.P on day zero and challenged with antigen (OVA)on days 13–18. These animals developed bronchial hyperresponsiveness(approximately two to three-fold over controls), and increased numbersof inflammatory cells including eosinphils in the airways asdemonstrated by elevated BAL cell counts as compared to controls (FIGS.20A and 20B top 2 panels). Many of the cells recruited to the airway asa result of this antigen challenge were eosinophils.

FIG. 22 panel 2 (bottom) shows a representative animal from thosepresensitized with OVA I.P on day zero, pretreated with polyclonalneutralizing antibodies to the murine IL-9 receptor (approximately 1mg/mouse I.P. in 0.5 ml of PBS), and challenged with antigen (OVA) ondays 13–18. This representative animal was protected from the responseto antigen. This response did not differ significantly bronchialresponsiveness, BAL cell counts from controls (FIGS. 20A and 20B top 2panels). These data demonstrate the potential effectiveness of treatingatopic allergy with antibodies to the IL-9 receptor.

Example 14

Murine Spleen Isolation and Culture

Mice were anesthetized and spleens were removed aseptically. Spleenswere minced with scissors and gently passed through a wire mesh(autoclaved) (#60 sieve). Cells were resuspended in 40 mls of RPMI-1640(GIBCO, BRL, Rockville, Md.), and spun for 5 min. at 250× G twice. Thepellet was resuspended in 10 mls of lysing butter to remove RBCs (4.15gm NH₄Cl, 0.5 gm KHCO₃; 019 g EDTA to 500 mls with ddH₂O). Cells wereincubated for about 5 minutes at 37° C. and 40 mls ofRPMI-FCS(RPMI-1640, 10% AFBS, 50 micromolar BME 2 mM glutamine,containing penicillin and streptomycin). These cells were spun again for5 minutes at 250× G and resuspended in 20 mls RPMI-FCS with or without 5micrograms/ml of concanavalin A (Sigma #C5275). IL-9 was assessed at 48hours in untreated splenocytes and after concanavalin A stimulation fromDBA/2J (D2) and C57BL/6J (B6) mice. IL-9 was amplified by RT-PCR (as setforth in Example 6), and probed with an IL-9 specific murine probe afterSouthern transfer. Southern blots were performed by “standard”techniques. Briefly, RT-PCR products were electrophoresed in 2% agarosegels. Gels were stained with ethidium bromide and photographed with aruler to determine molecular weight of DNA in southern blot. Gels werethen soaked in 0.5N NaOH for 30 minutes and neutralized in 0.5M Tris, pH7.0 for 30 minutes. DNA was transferred to zetaprobe (BioRAD) nylonmembrane by capillary transfer in 20× SSC overnight. The next day, themembrane was air dried, baked at 80° C. for 15 minutes and prehybridizedin 6× SSC and 0.1% SDS for 1 hour at 42° C. A kinase end-labelled ³²Poligonucleotide probe (5′-AATTACCTTATTGAAAATCTGAAG-3′) (SEQ ID NO: 44)was added to the hybridization solution plus 0.1 mg/ml sheared salmonsperm DNA and incubated overnight at 42° C. The next day, the filter waswashed in 3× SSC and 0.1% SDS at 37° C. for 30 minutes, and the filterwas exposed to film for 1 hour. FIG. 25 illustrates steady state levelsof IL-9 after 48 hours from each strain of mice. IL-9 was observed inunstimulated D2 (D2−) splenocytes, whereas no IL-9 was detectable in B6(B6−) mice. While there was a significant increase of IL-9 afterconcanavalin A stimulation in D2 (D2+) splenocytes, there was nodetectable IL-9 in B6 (B6+) mice despite concanavalin A treatment.

Example 15

Expression of Human Met117 IL-9 and Thr117 IL-9 in PBMCs SDS-PAGE andImmunoblot Analysis

After obtaining proteins isolated from human PBMC of healthy donorsinhibiting either the wild type (Thr117) or Met117-IL-9 genotypes as setforth in Example 13, SDS-PAGE was performed by the method of Laemmli(Laemmli U.K. (1970) Nature 227, 680–685) by using a 18% polyacrylamidegel in a mini-gel system (Xcell II vertical gel unit, Novex). Forimmunoblot analysis, the proteins separated by SDS-PAGE were transferredto nitrocellulose membranes by using the SD transblot transfer unit(Biorad) in 25 mM Tris-glycine buffer, pH 8.3, containing 15% methanol(Towbin et al., (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350–4354). Theunoccupied binding cites on the membrane were blocked by incubating for1 hour to overnight with 20 mM Tris-HCl buffer, pH 8.0, plus 0.05% tween20 (TBST) containing 5% dry milk. The membranes were then incubated with1:1000 dilution of goat anti-human IL-9 polyclonal antibody (R&DSystems) for 1 hour at room temperature. The membranes were washed withTBST and treated with 1:10,000 dilution of mouse anti-goat TgGconjugated with horseradish peroxidase for 1 hour. After washing withTBST, the bound antibodies were visualized by addition of the supersignal substrate chemiluminescence system (Pierce).

FIG. 23 demonstrates the expression of human IL-9 proteins from culturedPBMCs 48 hours after mitogen stimulation in individuals whose genotypeshave been determined by genomic analysis of the IL-9 gene. Lane 1 is aMet117 homozygote, lane 2 is a heterozygote Met117/Thr117, lane 3 is aThr117 homozygote. A single product of the approximate expected size (14kD) was seen in each individual PBMCs after mitogen stimulation. Thesedata demonstrate that both forms of the IL-9 protein are expressed andstable at steady state.

While the invention has been described and illustrated herein byreferences to various specific materials, procedures and examples, it isunderstood that the invention is not restricted to the particularmaterial combinations of material, and procedures selected for thatpurpose. Numerous variations of such details can be implied as will beappreciated by those skilled in the art.

REFERENCES

-   1. Gergen et al. (1992) The increasing problem of asthma in the    United States, Am. Rev. Respir. Dis. 146:823–824.-   2. Martin, (1985) Goodman and Gilman's The Pharmacologic Basis of    Therapeutics, Seventh Edition, MacMillan Publishing Company.-   3. Burrows et al. (1989) Association of asthma with serum IgE levels    and skin-test reactivity to allergens, N. Engl. J. Med. 320:271–277.-   4. Clifford et al. (1987) Symptoms, atopy, and bronchial response to    methacholine in parents with asthma and their children, Arch. Dis.    in Childhood 62:66–73.-   5. Gergen (1991) The association of allergen skin test reactivity    and respiratory disease among whites in the U.S. population, Arch.    Intern. Med. 151:487–492.-   6. Burrows et al. (1992) Relationship of bronchial responsiveness    assessed by methacholine to serum IgE, lung function, symptoms, and    diagnoses in 11-year-old New Zealand children, J. Allergy Clin.    Immunol. 90:376–385.-   7. Johannson et al. (1972) The clinical significance of IgE, Prog.    Clin. Immunol. 1:1–25.-   8. Sears et al. (1991) Relation between airway responsiveness and    serum IgE in children with asthma and in apparently normal    children, N. Engl. J. Med. 325(15):1067–1071.-   9. Halonen et al. (1992) The predictive relationship between serum    IgE levels at birth and subsequent incidences of lower respiratory    illnesses and eczema in infants, Am. Rev. Respir. Dis. 146:666–670.-   10. Marsh et al. (1982) The epidemiology and genetics of atopic    allergy, N. Engl. J. Med. 305:1551–1559.-   11. Hopp et al. (1988) Bronchial reactivity pattern in nonasthmatic    parents of asthmatics, Ann. Allergy 61:184–186.-   12. Hopp et al. (1990) The presence of airway reactivity before the    development of asthma, Am. Rev. Respir. Dis. 141:2–8.-   13. Ackerman et al. (1994) Detection of cytokines and their cell    sources in bronchial biopsy specimens from asthmatic patients:    relationship to atopic status, symptoms, and level of airway    hyperresponsiveness, Chest 105:687–696.-   14. Hamid et al. (1991) Expression of mRNA for interleukin-5 in    mucosal bronchial biopsies from asthma, J. Clin. Invest.    87:1541–1546.-   15. Djukanovic et al. (1990) Mucosal inflammation in asthma, Am.    Rev. Respir. Dis. 142:434–457.-   16. Robinson et al. (1992) Predominant TH2-like bronchoalveolar T    lymphocyte population in atopic asthma, N. Engl. J. Med.    326:298–304.-   17. Robinson et al. (1993) Prednisolone treatment in asthma is    associated with modulation of bronchoalveolar lavage cell    interleukin-4, interleukin-5, and interferon-cytokine gene    expression, Am. Rev. Respir. Dis. 148:401–406.-   18. Robinson et al. (1993) Relationship among numbers of    bronchoalveolar lavage cells expressing messenger ribonucleic acid    for cytokines, asthma symptoms, and airway methacholine    responsiveness in atopic asthma, J. Allergy Clin. Immunol.    92:397–403.-   19. Sears et al. (1991) Relation between airway responsiveness and    serum IgE in children with asthma and in apparently normal    children, N. Engl. J. Med. 325:1067–1071.-   20. Burrows et al. (1992) Relationship of bronchial responsiveness    assessed by methacholine to serum IgE, lung function, symptoms, and    diagnoses in 11-year-old New Zealand children, J. Allergy Clin.    Immunol. 90:376–385.-   21. Clifford et al. (1987) Symptoms, atopy, and bronchial response    to methacholine in parents with asthma and their children, Arch.    Dis. Childhood 62:66–73.-   22. O'Connor et al. (1989) The role of allergy and nonspecific BHR    in the pathogenesis of COPD, Am. Rev. Respir. Dis. 140:225–252.-   23. Cogswell et al. (1982) Respiratory infections in the first year    of life in children at the risk of developing atopy, Brit. Med. J.    284:1011–1013.-   24. Boushey et al. (1980) BHR, Am. Rev. Respir. Dis. 121:389–413.-   25. Cookson et al. (1989) Linkage between immunoglobin E responses    underlying asthma and rhinitis and chromosome 11q, Lancet.    1:1292–1295.-   26. Moffatt et al. (1992) Factors confounding genetic linkage    between atopy and chromosome 11q, Clin. Exp. Allergy 22:1046–1051.-   27. Amelung et al. (1992) Atopy, asthma and bronchial    hyperresponsiveness: Exclusion of linkage to markers on chromosome    11q and 6p, Clin. Exper. Allergy 22:1077–1084.-   28. Rich et al. (1992) Genetic evidence of atopy in three large    kindreds: no evidence of linkage to D11S97, Clin. Exp. Allergy    22:1070–1076.-   29. Lympany et al. (1992a) Genetic analysis using DNA polymorphism    of the linkage between chromosome 11q13 and atopy and BHR to    methacholine, J. Allergy Clin. Immunol. 89:619–628.-   30. Lympany et al. (1992b) Genetic analysis of the linkage between    chromosome 11q and atopy, Clin. Exp. Allergy 22:1085–1092.-   31. Hizawa et al (1992) Lack of linkage between atopy and locus    11q13, Clin. Exp. Allergy 22:1065–1069.-   32. Sanford et al. (1993) Localization of atopy and b subunit of    high-affinity IgE receptor (FceR1) on chromosome 11q, Lancet.    341:332–334.-   33. Shirakawa et al. (1994) Association between atopy and variants    of the beta subunit of the high-affinity immunoglobulin E receptor,    Nature Genetics 7:125–130.-   34. Marsh et al. (1974) Genetic control of basal serum    immunoglobulin E level and its effect on specific reaginic    sensitivity, Proc. Natl. Acad. Sci. USA 71:3588–3592.-   35. Gerrard et al. (1978) A genetic study of IgE, Am. J. Hum. Genet.    30:46–58.-   36. Meyers et al. (1987) Inheritance of serum IgE (basal levels) in    man, Am. J. Hum. Genet. 41:51–62.-   37. Meyers et al. (1982) A genetic study of total IgE levels in the    Amish, Hum. Hered. 32:15–23.-   38. Martinez et al. (1994) Evidence for mendelian inheritance of    serum IgE levels in Hispanic and Non-hispanic white families, Am. J.    Hum. Genet. 55:555–565.-   39. Blumenthal et al. (1981) Genetic transmission of serum IgE    levels, Am. J. Med. Genet. 10:219–228.-   40. The Genome Data Base. The Welch Library, The Johns Hopkins    Medical Institutions, Baltimore, Md., USA.-   41. Marsh et al. (1994) Linkage analysis of IL4 and other chromosome    5q31.1 markers and total serum immunoglobulin E concentrations,    Science 264:1152–1156.-   42. Meyers et al. (1994) Evidence for a locus regulating total serum    IgE levels mapping to chromosome 5, Genomics 23:464–470.-   43. Doull et al. (1996) Allelic association of makers on chromosome    5q and 11q with atopy and bronchial hyperresponsiveness, Am. J.    Respir. Crit. Care Med. 153:1280–1284.-   44. Ott (1991) Analysis of human genetic linkage. Baltimore, Md.:    The Johns Hopkins University Press.-   45. Renauld et al. (1993) Interleukin-9, Int. Rev. Exp. Pathology    134A:99–109.-   46. Renauld et al. (1995) Interleukin-9 and its receptor:    involvement in mast cell differentiation and T cell oncogenesis, J.    Leukoc. Biol. 57:353–360.-   47. Hultner et al. (1989) Thiol-sensitive mast cell lines derived    from mouse bone marrow respond to a mast cell growth-enhancing    activity different from both IL-3 and IL-4, J. Immunol.    142:3440–3446.-   48. Dugas et al. (1993) Interleukin-9 potentiates the    interleukin-4-induced immunoglobulin (IgG, IgM and IgE) production    by normal human B lymphocytes, Eur. J. Immunol. 23:1687–1692.-   49. Petit-Frere et al. (1993) Interleukin-9 potentiates the    interleukin-4-induced IgE and IgG1 release from murine B    lymphocytes, Immunology 79:146–151.-   50. Behnke et al. (1993) Immunological relationships during primary    infection with Heligmosomoides polygyrus (Nematospiroides dubius):    downregulation of specific cytokine secretion (IL-9 and IL-10)    correlates with poor mastocytosis and chronic survival of adult    worms, Parasite Immunol. 15:415–421.-   51. Gessner et al. (1993) Differential regulation of IL-9 expression    after infection with Leischmania major in susceptible and resistant    mice, Immunobiology 189:419–435.-   52. Renauld et al. (1992) Expression cloning of the murine and human    interleukin 9 receptor cDNAs, Proc. Natl. Acad. Sci. 89:5690–5694.-   53. Chang et al. (1994) Isolation and characterization of the Human    interleukin-9 receptor gene, Blood 83:3199–3205.-   54. Renauld et al. (1990) Human P40/IL-9. Expression in activated    CD4+ T cells, Genomic Organization, and Comparison with the Mouse    Gene, J. Immunol. 144:4235–4241.-   55. Kelleher et al. (1991) Human interleukin-9: genomic sequence,    chromosomal location, and sequences essential for its expression in    human T-cell leukemia virus (HTLV-1-transformed human T cells, Blood    77:1436–1441.-   56. Houssiau et al. (1995) A cascade of cytokines is responsible for    IL-9 expression in human T cells. Involvement of IL-2, IL-4, and    IL-10, J. Immunol. 154:2624–2630.-   57. Miyazawa et al. (1992) Recombinant human interleukin-9 induces    protein tyrosine phosphorylation and synergizes with steel factor to    stimulate proliferation of the human factor-dependent cell line,    MO7e, Blood 80:1685–1692.-   58. Yin et al. (1994) JAK1 kinase forms complexes with interleukin-4    receptor and 4PS/insulin receptor substrate-1-like protein and is    activated by interleukin-4 and interleukin-9 in T lymphocytes, J.    Biol. Chem. 269:26614–26617.-   59. Renauld et al. (1992) Expression cloning of the murine and human    interleukin 9 receptor cDNAs, Proc. Natl. Acad. Sci. 89:5690–5694.-   60. Chang et al. (1994) Isolation and characterization of the Human    interleukin-9 receptor gene, Blood 83:3199–3205.-   61. Kreitman et al. (1994) Site-specific conjugation to interleukin4    containing mutated cysteine residues produces interleukin 4-toxin    conjugates with improved binding and activity, Biochemistry    33:11637–11644.-   62. Simoncsits et al. (1994) Deletion mutants of human interleukin 1    beta significantly reduced agonist properties: search for the    agonist/antagonist switch in ligands to the interleukin 1 receptors,    Cytokine 6:206–214.-   63. Zav'yalov et al. (1992) Nonapeptide corresponding to the    sequence 27–35 of the mature human IL-2 efficiently competes with    rIL-2 for binding to thymocyte receptors, Immunol. Lett. 31:285–288.-   64. Chu et al. (1992) Glycophorin A interacts with interleukin-2 and    inhibits interleukin-2-dependent T-lymphocyte proliferation, Cell    Immunol. 145:223–239.-   65. Alexander et al. (1992) Trial of cyclosporin in    corticosteroid-dependent chronic severe asthma, Lancet. 339:324–328.-   66. Morely (1992) Cyclosporin A in asthma therapy: a pharmacological    rationale, J. Autoimmun. 5 Suppl. A:265–269.-   67. Lander et al. (1989) Mapping Mendelian factors underlying    quantitative traits using RFLP linkage maps, Genetics 121:185–199.-   68. Soller et al. (1976) On the power of experimental designs for    the detection of linkage between maker loci and quantitative loci in    crosses between inbred lines, Theor. Appl. Genet. 47:35–39.-   69. Kvaloy et al. (1994) The sequence organization of the long arm    pseudoautosomal region of the human sex chromosomes, Hum. Mol.    Genet. 3:771–778.-   70. Freije et al. (1992) Identification of a second pseudoautosomal    region near the Xa and Ya telomers, Science 258:1784–1787.-   71. Weber et al. (1989) Abundant class of human DNA polymorphisms    which can be typed using the polymerase chain reaction, Am. J. Human    Genet. 44:388–396.-   72. Saiki et al. (1988) Primer-directed enzymatic amplification of    DNA with a thermostable DNA polymerase, Science 239:487–491.-   73. Sheffield et al. (1993) The sensitivity of single-strand    conformation polymorphism analysis for the detection of single base    substitutions, Genomics 16:325–332.-   74. Orita et al. (1989) Rapid and sensitive detection of point    mutations and DNA polymorphisms using the polymerase chain reaction,    Genomics 5:874–879.-   75. Sarkar et al. (1992) Dideoxy fingeringprint (ddF): A rapid and    efficient screen for the presence of mutations, Genomics 13:441–443.-   76. Cotton (1989) Detection of single base changes in nucleic acids.    Biochem. J. 263(1):1–10.-   77. Schwengel et al. (1993) Linkage mapping of the human thromboxane    A2 receptor (TBXA2R) to chromosome 19 p13.3 using transcribed 3′    untranslated DNA sequence polymorphisms, Genomics 18:212–215.-   78. SAGE, Statistical Analysis for Genetic Epidemiology (1992).    Release 2.1. Computer program package available from the Department    of Biometry and Genetics, LSU Medical Center, New Orleans, La.-   79. Postma et al. (1995) Genetic Susceptibility to Asthma: Bronchial    Hyperresponsiveness Coinherited with a Major Gene for Atopy, N.    Engl. J. Med. 333:894–900.-   80. Xu et al. (1995) Evidence for two-unlinked loci regulating serum    total IgE levels, Am. J. Hum. Genet. 57:425–430.-   81. Meyers (1995) Two locus segregation and linkage analysis for    total serum IgE levels, Clin. Exp. Allergy 25:113–115.-   82. Bleecker et al. (1995) Evidence for linkage of total serum IgE    and bronchial hyperresponsiveness to chromosome 5q: a major    regulatory locus important in asthma, Clin. Exp. Allergy 25:84–88.-   83. Panhuysen et al. (1995) Evidence for a susceptibility locus for    asthma mapping to chromosome 5q, J. Investig. Med. 43:281A.-   84. Levitt et al. (1995) Linkage homology for bronchial    hyperresponsiveness between DNA markers on human chromosome 5q31-q33    and mouse chromosome 13, Clin. Exp. Allergy 25:61–63.-   85. Yang et al. U.S. Pat. No. 5,414,071, Human cytokine IL-9 (May 9,    1995).-   86. Alms et al. PCT/US95/04094 (WO 95/27052), Human interleukin    variants generated by alternative splicing.-   87. Thomson (1994) Cytokine handbook, Academic Press.-   88. Martinati et al. (1996) Affected sib-pair and mutation analyses    of the high affinity IgE receptor beta chain locus in Italian    families with atopic asthmatic children, Am. J. Respir. Crit. Care    Med, 153:1682–1685.-   89. Kauvar (1996) Peptide mimetic drugs: A comment on progress and    prospects, Nature Biotechnology 14:709.

1. An isolated human IL-9 mutein peptide comprising an amino acidsubstitution at residue 117 of human IL-9 wild-type amino acid sequence.2. The isolated IL-9 mutein peptide of claim 1 wherein the peptide is anIL-9 antagonist.
 3. The isolated IL-9 mutein peptide of claim 1 whereinsaid substitution at residue 117 is from threonine to an amino acidselected from the group consisting of alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan and methionine.
 4. Theisolated IL-9 mutein peptide of claim 1 wherein said substitution atresidue 117 is threonine to methionine.
 5. The isolated IL-9 muteinpeptide of claim 1 wherein said peptide comprises the amino acidsequence of SEQ ID NO:
 27. 6. A fusion protein comprising the isolatedIL-9 mutein peptide of claim 1 and a second protein.
 7. The fusionprotein of claim 6 wherein the second protein comprises the amino acidsequence SEQ ID NO:
 45. 8. The IL-9 mutein peptide of claim 1 whereinsaid peptide inhibits the proliferative response of Mo7E cells to IL-9with an IC₅₀ value of about 1 to about 100 micrograms per mil.
 9. Amethod of treating atopic allergy in a patient in need of such treatmentcomprising administering to said patient a therapeutically effectiveamount of the IL-9 mutein peptide of claim
 1. 10. The method of claim 9wherein the atopic allergy is asthma.
 11. The method of claim 9 whereinthe atopic allergy is associated with one or more symptoms in thepatient selected from the group consisting of reversible airflowobstruction, bronchial hyperresponsiveness (BHR), rhinitis, urticaria,inflammatory bowel disease and eczema.
 12. The method of claim 9 whereinthe patient is human.